Plasmodium falciparum was determined in patients seeking medical care primarily for non-febrile conditions in six departments in Haiti using a newly designed qRT-PCR-based assay.Public health measures are poised for transition from malaria control to malaria elimination on the island of Hispaniola. Assessment of the reservoir of asymptomatic infections from which acute malaria cases may derive is critical to plan and evaluate elimination efforts. Current field technology is ill suited for detecting sub-microscopic infections, thus highly sensitive survey methods capable of detecting virtually all infections are needed. In this study the prevalence of infection with P. falciparum infections in the population: malaria rapid diagnostic test (RDT) designed to detect histidine-rich protein 2 (HRP2), thick smear microscopy, and a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based upon the small sub-unit ribosomal RNA. The limit of detection of the qRT-PCR assay utilized was 0.0003 parasite/µL of blood. Venous blood was obtained from a total of 563 subjects from six departments in Haiti, all of whom were seeking medical attention without complaints consistent with malaria. Each subject was questioned for knowledge and behaviour using demographic and epidemiological survey to identify risk factors for disease transmission.Three different methods of parasite detection were compared to assess their utility in approximating the prevalence of Among the 563 samples tested, ten and 16 were found positive for malaria by RDT and microscopy, respectively. Using the qRT-PCR test to assess the infection status of these subjects, an additional 92 were identified for a total of 108. Based upon the qRT-PCR assay results, a wide variation in prevalence of infection in asymptomatic subjects was seen between geographic locations ranging from 4–41 %. The prevalence of infection was highest in the Grand Anse, Nord and Sud-Est Departments, and demographic data from questionnaires provide evidence for focal disease transmission.The qRT-PCR assay is sufficiently sensitive to identify an unexpectedly large number of asymptomatic, submicroscopic infections. Identifying and clearing these infections presents a significant challenge to both control and elimination efforts, but the qRT-PCR assay offers a reliable method to identify them. Anopheles albimanus is the predominant vector transmitting malaria on Hispaniola, and the causative agent is Plasmodium falciparum [Hispaniola is the only Caribbean island where malaria is endemic, and the greatest burden is borne by Haiti, located on the western half of the island . Anophellciparum . The natlciparum , 4, the lciparum . Malarialciparum , 7. Offilciparum .P. falciparum appears to be the single agent of malaria [Since the vast majority of malaria cases on Hispaniola occur in Haiti, it should remain the focus of malaria control and future eradication efforts , 8, 9. E malaria , 9, 11.  malaria . All of  malaria .Rapid diagnostic tests have been identified as a key component of the current control strategy to prepare for eradication, thus keeping microscopy as only a confirmatory secondary test . RDTs haP. falciparum to persist in an asymptomatic host for more than a decade, making these infections potentially very important in elimination efforts [Moving beyond the malaria control phase to the elimination phase, it is suspected that in comparison to the whole population carrying malaria parasites, the febrile cases represent only the tip of the iceberg. The sub-population harbouring asymptomatic, sub-microscopic infections remains obscure. A recent review has described the potential for  efforts . Further efforts , 17. Des efforts . AdditioFor the past five years malaria transmission in Haiti has been fairly stable, with a promising low level of overall transmission. However, in Africa and other areas around the world  where the transmission levels drop significantly, substantial numbers of sub-microscopic carriers remain, enabling the parasite to restart transmission when conditions are right –22. In TThis study was conducted in accordance with institutional review board guidelines and requirements of the University of Florida and the ethical review board of the Haitian MSPP after obtaining all permits and approvals . Subjects were recruited through a cross-sectional sampling of reproductive age women across Haiti in gynecology and maternity clinics. Study team collaborated with NGOs and government dispensaries to enrol women for free malaria screening. Each participant during the waiting time for enrolment was asked to answer a demographic survey in Haitian Creole. The questionnaire included general demographic and behaviour questions such as gender, age, geographic residency, history of travel, sleeping pattern (outdoor and/or indoor), presence of window screens, use of insecticide-treated bed nets, presence of bed nets whether treated or not, and history of malaria transmission for the participant and household members. Each questionnaire was labelled with a unique identifying number that matched the consent form and sample to provide de-identified data for analysis.Study sites shown in Fig. P. falciparum small sub-unit ribosomal RNA type A [Plasmodium spp. infecting humans  along with human small sub-unit rRNA using ClustalW2 software, and selecting specific primer pairs and probe using Integrated DNA Technologies, Inc. software. Care was taken to assure that the primers and probe would detect the small sub-unit rRNA, which is present predominantly in P. falciparum blood stage parasites. The following primers were selected for use in the qRT-PCR assay employed here: forward (5-GATACCGTCGTAATCTTAACCATAAAC-3), reverse (5-AAGGTACTGAAGGAAGCAATCT-3) and probe (6-FAM-ACACTTTCATCCAACACCTAGTCGGC-BHQ-1). The specificity of the primers was confirmed experimentally with RNA from P. falciparum clone 3D7 and RNA from uninfected human blood.A set of primers and a probe were designed to develop a highly sensitive molecular test to detect the presence of A type A , 25. TheTotal RNA was extracted from 800 µL of the mixture of blood preserved in DRS using the Quick-RNA™ MiniPrep Kit following the RNA isolation from red blood cells protocol (Zymo Research) in a PCR workstation. The RNA was eluted in a volume of 50 µL of which 5 µL was used in the qRT-PCR reaction. Total nucleic acid extraction was also performed from blood samples in DRS using the same kit, but following a modification recommended by the manufacturer for obtaining total nucleic acids. Total nucleic acids were also eluted in 50 µL of which 5 µL was used in subsequent qPCR and qRT-PCR reactions.The qRT-PCR was conducted using the Express One-Step SuperScript qRT-PCR Kit (Invitrogen) following manufacturer’s instructions with a final concentration of 0.4 µM of each primer and 0.2 µM for the probe in a 20 µL reaction. The PCR master mix preparation was conducted in a separate PCR work station. Amplification and real-time measurements were performed on a CFX96 Touch ™ Real-Time PCR Detection system (Bio-Rad). Reaction conditions were as follows: 20 min at 58 °C for reverse transcription step, one cycle of 2 min at 95 °C for Taq DNA Polymerase activation and 40 cycles of 95 °C for 15 s, 60 °C for 1 min for annealing and extension of amplified product. Every PCR run was conducted with two negative controls and one positive control. Samples were tested in separate batches where those with positive RDT results were extracted and tested separate from the ones with negative RDTs to reduce the possibility of cross contamination.P. falciparum 3D7 free of mature parasites and most culture debris was prepared as follows to obtain parasitized cells containing ring stage parasites, the developmental stage expected in the peripheral blood of infected patients. A fresh, semi-synchronous culture was prepared by the sorbitol method [5 parasites/ml.A synchronous culture of l method  and matul method . The mat5 parasites/ml and serially diluted in RNase/DNase free water in intervals of tenfold dilution with the final dilution equivalent to 0.1 parasites/ml of blood. All dilutions were treated with RNase inhibitor using RNase Out (Life Technologies) with a final concentration of 1 unit per µL. The same blood sample having 105 parasites/ml was serially diluted as well in un-infected blood at intervals of ten-fold to reach a concentration of 10 parasites/ml of blood, then at intervals of two-fold dilution until reaching a concentration of 0.3 parasites/ml of blood. Infected blood (200 µL) was then mixed with 600 µL of DRS and RNA was extracted from all 800 µL as described above. RNA was eluted in a volume of 50 µL, of which 5 µL was used as a template in each qRT-PCR reaction.A standard curve was prepared from RNA extracted from the initial sample containing 10P. falciparum parasites. After stratification for demographic location by department, simple logistic regression models were used to determine the likelihood of carrying P. falciparum parasites using survey questions in Stata V.11 . Questions included items relating to participant knowledge of malaria, risk factors for malaria infection, demographic characteristics, and human mobility characteristics. Statistical significance was determined at an alpha level of 0.05 in all logistic regression models.Demographic and survey items were used to assess differences in the likelihood of being an asymptomatic carrier of P. falciparum was 0.0003 parasitized red cells per µL of whole blood (0.3 per mL)  Fig. a. In tesmL) Fig. b. AssaysmL) Fig. a was usemL) Fig. c, since mL) Fig. c. ResultOverall 3.0 % of samples tested were positive for malaria by microscopic analysis, 2.4 % by RDT and 19.1 % by qRT-PCR. However, a large variation in prevalence of infection was detected across departments. Departments having a low prevalence of malaria infections  are Artibonite, Central Plateau and Ouest. High prevalence  was observed in the Nord, Sud-Est and Grand-Anse Departments. Thus, a large reservoir of asymptomatic, sub-microscopic carriers is present in various locations across the peninsula interspersed with departments with very low prevalence.No significant risk factors emerged from the regression analysis for insecticide usage nor did sleeping outdoors during morning or night correlate with a positive qRT-PCR result. However, travelling outside the screening area within the last month was shown to be a protective factor with odds ratio of 0.53 and (P = 0.004). Those who were qRT-PCR-positive were more likely to know about malaria as a disease and its mode of transmission, with an odds ratio of 1.79 (P = 0.009). The odds of being positive for malaria by qRT-PCR when a household member was also infected were 2.2 times higher than for those who did not live near a malaria-infected family member, and this relationship was found to be statistically significant with a (P = 0.002). Lastly, the odds of being positive by qRT-PCR with a history of previous infection was 2.1 times higher than for those who were self-identified as not previously having been infected (P = 0.003). A summary of results for the demographic questionnaire is presented in Table A survey method capable of identifying virtually every individual infected with the parasite will be crucial in the malaria elimination phase. It will allow making a reliable estimate of the proportion of sub-microscopic infections which must be factored in when developing intervention plans , and, secondly to test the efficacy of intervention measures after implementation to evaluate success . PCR hasP. falciparum with a limit of detection of 0.0003 parasites/µL of blood exceeds this threshold by an order of magnitude. Comparing the qRT-PCR assay to qPCR on total nucleic acids using the same primer sets and reaction conditions, excluding the reverse transcription step, Ct values for qRT-PCR were an average of 14 lower than for qPCR for the same sample. The large number of rRNA molecules in each parasite (>104) [The qRT-PCR assay described here has the potential to detect blood stage infections from the first emergence of the parasite into the blood from the liver throughout the course of blood stage infection, even during what appears to be an asymptomatic, chronic phase. A single exo-erythrocytic (EE) stage schizont releases merosomes into the blood stream, which ultimately rupture releasing tens of thousands of merozoites. Assuming an average blood volume of 5 L and a single exoerythrocytic stage schizont releasing merozoites, then two or more infected red cells may be expected per ml of blood to initiate the asexual growth phase. To detect this parasitaemia, the test must have a limit of detection equal to or lower than 0.004 parasites/µL of blood. The qRT-PCR targeting the small ribosomal RNA of e (>104) , their pIn order for Haiti to reach the pre-elimination phase of malaria, the number of active cases must be less than one per 1000 population at risk in preparation for a reduction to zero locally acquired cases. Haiti would have to maintain zero locally acquired cases for at least 3 years prior to becoming certified malaria-free country by WHO . The curComparatively high malaria prevalence has been reported previously in the Grande Anse, Nord and Sud-Est Departments , 18, howAn. albimanus, an outdoor-biting mosquito with a preference for blood meals at dawn and dusk [In spite of the availability of bed nets to 64 % of the population, no statistical significance for their proposed role in protecting individuals from infection was found. This finding is consistent with the fact that the main vector for malaria transmission in Haiti is and dusk . The surThere is a large variation in malaria prevalence among study sites, and these results suggest that using this highly sensitive qRT-PCR based survey tool would facilitate accurately mapping the reservoirs of malaria across the country. Given that samples obtained in a field setting are stable for transportation to established laboratories for rapid completion of the assay, the results can be obtained in a timely fashion for mapping purposes and for assessing effectiveness of elimination efforts. Obtaining such data will be crucial to effectively guiding the utilization of resources available for elimination and evaluating success.The findings of this study demonstrate the superior efficacy of the qRT-PCR as a surveillance tool in detecting malaria infections, including submicroscopic infections, as compared to other available tools frequently employed including PCR. DRS provides a highly useful preservative for parasite nucleic acids in field conditions, making this assay feasible for use under field conditions in underdeveloped countries by allowing successful transportation of stabilized samples to established laboratories for analysis. The study identified a large number of sub-microscopic infections in Haiti that were not previously described. Plans for malaria elimination on Hispaniola must account for the large number of asymptomatic, sub-microscopic infections present in Haitians in regions where malaria transmission is found.


Plasmodium species and transmitted by anopheline mosquitoes. It occurs in single and mixed infections. Mixed infection easily leads to misdiagnosis. Accurate detection of malaria species is vital. Therefore, the study was conducted to determine the level of mixed infection and misdiagnosis of malaria species in the study area using SYBR Green I-based real time PCR.Malaria is caused by five The study was conducted in seven health centres from North Gondar, north-west Ethiopia. The data of all febrile patients, who attended the outpatient department for malaria diagnosis, from October to December 2013, was recorded. Dried blood spots were prepared from 168 positive samples for molecular re-evaluation. Parasite DNA was extracted using a commercial kit and Plasmodium species were re-evaluated with SYBR Green I-based real time PCR to detect mixed infections and misdiagnosed mono-infections.Plasmodium falciparum, 553 (30.68%) Plasmodium vivax and 33 (1.8%) mixed infections of both species were recorded. The result showed high prevalence of P. falciparum and P. vivax, but very low prevalence of mixed infections. Among 168 samples collected on dried blood spot 7 (4.17%) were P. vivax, 158 (94.05%) were P. falciparum and 3 (1.80%) were mixed infections of both species. After re-evaluation 10 (5.95%) P. vivax, 112 (66.67%) P. falciparum, 21 (12.50%) P. falciparum + P. vivax mixed infection, and 17 (10.12%) Plasmodium ovale positive rate was recorded. The re-evaluation showed high level of mixed infection, and misdiagnosis of P. ovale and P. vivax.Among 7343 patients who were diagnosed for malaria in six study sites within the second quarter of the Ethiopian fiscal year (2013) 1802 (24.54%) were positive for malaria parasite. Out of this, 1,216 (67.48%) P. falciparum prevalence is higher than P. vivax in the study area. The results, obtained from SYBR Green I-based real time PCR, indicated that the diagnosis efficiency of microscopy is very low for species-specific and mixed infection detection. Therefore, real time PCR-based species diagnosis should be applied for clinical diagnosis and quality control purposes in order to prevent the advent of drug resistant strains due to misdiagnosis and mistreatment.The result shows that  Plasmodium, and is transmitted by anopheline mosquitoes. Plasmodium parasite has five species affecting humans. These are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. These species cause approximately 225 million infections and over 600,000 deaths per year. Among them, P. falciparum is the most prevalent and common malaria species worldwide, especially in Africa. It causes the most severe form of the disease and is responsible for over 90% of the malaria patients’ death[Malaria, together with other infectious diseases such as tuberculosis and HIV, is an important cause of morbidity and mortality. Malaria is caused by the parasite s’ death–3.Clinical diagnosis of Plasmodium species by microscopy is not precise. However, it is still the basis of therapeutic care and plays a key role in the diagnosis of febrile patients in malaria endemic areas. Laboratories in malaria-endemic area needs accurate and precise diagnosis of mono-infection and mixed species infections in order to assure proper treatment decision. This helps to prevent the advent of drug resistant parasite population. Reasonable malaria diagnosis and treatment is essential to avoid non-target effects and to save cost on alternative drugs. Accurate and effective diagnosis is the only way of assuring rational treatment and therapy.P. falciparum infection is influenced by its parasite density. Parasite density of infections by non-falciparum Plasmodium species is usually low compared to P. falciparum[P. falciparum parasites makes the observation difficult to differentiate other species[Microscopic observation of lciparum. Therefo species.P. falciparum and P. vivax in a single human host suppress each other. Plasmodium falciparum can suppress P. vivax parasitaemia by interspecies inhibition. Severity of malaria, in mixed species infection, depends on whether it is a P. falciparum or a P. vivax super infection. Plasmodium vivax super infection over an existing P. falciparum infection leads to the rise of P. falciparum parasitaemia and causes severe malaria. In contrast, P. falciparum super infection over an existing P. vivax infection reduces P. falciparum parasitaemia. Therefore, it prevents the development of severe malaria[Mixed species infection can not only complicate diagnosis, but also alter the severity and morbidity of the disease. Co-existence of  malaria.P. falciparum and P. vivax in Ethiopia and the different levels of effectiveness of the anti-malarial drugs against the malaria parasite species demand administration of the right drug to control morbidity and mortality. In the Ethiopian setting, therefore, the current ongoing efforts to increase access to diagnostic services, including the use of appropriate rapid diagnostic tests (RDTs) are expected to have a significant contribution[P. falciparum and P. vivax mixed infections occur with high frequency[The co-existence of ribution. In malarequency.Mixed infection incidence is less than the prevalence of individual species and it is also seasonal. Malaria transmission occurs more in wet season than dry season. The seasonal variation is due to the spread of various mosquito species in wet season. The spread of various mosquitoes in malarious area causes high incidence of both single and mixed infections in wet season than dry season. RelativP. vivax infection, treatment of P. vivax increases P. falciparum parasitaemia. Mixed-species infections increase the possibility of anti-malarial drug resistance. Hence, a drug-resistant population of Plasmodium parasites will emerge[If mixed-species malaria is misdiagnosed as a single l emerge. Therefol emerge, 10. It Microscopy is a “gold standard” method which remains the most appropriate method for malaria diagnosis in resource-limited area. Most microscopists who see one species might not look for another and misclassification is common due to lack of microscopic skills to differentiate morphological variation within and between species, 11. It P. falciparum and P. vivax using real-time PCR.PCR is a better tool for measuring Plasmodium prevalence and detection of both asymptomatic and symptomatic Plasmodium parasites than microscopy. Submicroscopic or low level parasites may not be detected by the conventional microscopy. Molecul036′N and longitude of 37028′E and 729 km away from the capital Addis Ababa. The district covers an area of 1,270 km2 with a total population of about 263,000. The area is endemic to malaria in which P. falciparum and P. vivax species are commonly reported from microscope diagnosis of febrile patients.The hospital is located in Gendawuha district which is found at a latitude of 12058′N and longitude of 36012′E with an elevation of 685 meter above sea level and approximately 950 km away from the capital Addis Ababa. The area has approximate population of 5,502. The two areas have severe malaria transmission seasons usually October to December . Six clinics  and one hospital  were used as a study site to collect samples from outpatients.P. falciparum and/or P. vivax positive during health service consultation were asked for consent by health professionals to be part of the study. Finger pricks were done by health professionals for those patients who consented to participate in the study.Patients who attended the health centres during study period were recruited for the study. Participants who were between six month and seventy years old were included in the study. Infants, under six months of age and pregnant women were excluded. Patients who were reported as P. vivax microscopically . The spots were properly labelled and air dried. Silica gel was added inside each plastic bag to prevent humidity. In order to avoid cross contamination one plastic bag was used for one filter paper. The cards were collected in sealed plastic bag and transported to Kenya Medical Research Institute (KEMRI) for molecular analysis.After finger pricking, two spots of blood samples were prepared on Whatman 3MM filter paper from each study participant. The blood spots were prepared by dropping three to four blood drops per spot on a piece of filter paper. In total, 168 samples were collected from all study sites . From the total samples three samples were mixed infections  and seven were reported as P. falciparum and P. vivax single infection as well as mixed infection of both species as it was diagnosed by microscopy. During this data collection, the total number of blood film made for malaria microscopic diagnosis, Plasmodium falciparum single infection, P. vivax single infection and mixed infections of both species were recorded from all the six institutions. Unlike the six health centres, the hospital provided only the total of P. falciparum single infection records, although there was no exclusion factor or criteria for the hospital to consider only P. falciparum infection. Secondary data was collected in order to determine the prevalence level of P. falciparum and P. vivax infection in the six health centres as it was diagnosed by microscopy. Due to its severity, P. falciparum single infection prevalence level was considered alone in all the study sites.In addition to blood samples collected on filter paper, secondary data was collected from each study site. The data was collected for the second quarter malaria report of the Ethiopian fiscal year (2013) using the report format used by each health centres. This data includes The research proposal was submitted to the University of Gondar Natural and Computational Science College Ethical Committee for Ethics approval. The Committee Approved the proposed research is Ethical after thorough consideration of the proposed research. Consent form was prepared for study participants. It was explained for participants and parents/guardians in their mother tongue about the purpose of the study before they were being participating in the study. Those who were not consented had withdrawn from participation. In agreement with the clinics and the hospital the cost of drug for treatment of positive patients were covered by the clinics and the hospital with follow up of health professionals.A piece of dried blood spot of approximately 2 mm-3 mm in size, was cut out with sterile scissors. The pieces were placed in sterile 1.5 ml extraction tubes using flamed forceps. High pure PCR template preparation kit  was used to extract parasite DNA from dried blood spots (DBS). The extraction was done according to manufacturer’s instruction. The extracted DNA was stored in -20°C freezer until used for PCR.TM qPCR Master mix and Exicycler 96TM (Bioneer South Korea). ExicyclerTM version 3.0 Software was used for programming the Exicycler Thermal Block (Exicycler 96) and data analyses.Two-step PCR was done using AccuPower 2x GreenStarConsensus primers were used to amplify a species-specific region of the multi-copy 18S rRNA gene. The 18S rRNA gene was used as a target since it contains both highly conserved and variable regions for each of Plasmodium species, and at least five copies of the gene are dispersed on separate chromosomes of the Plasmodium. These cP. falciparum laboratory cultured strain 3D7, and microscopically-positive P. vivax were used as control group. Microscopically P. vivax positive samples were reconfirmed before being used as a control, with another PCR assay in which P. vivax specific published primer pair Pvr47-F (5′-CTT ATT TTCCGC GTA ACA ATG-3′) and Pvr47-R (5′-CAA ATG TAG CAT AAA AAT CTA AG-3′) were used for conformation[The amplification was done at 50 μl reaction volume. 25 μl master mix mentioned above, 2 μl (10 pmole) forward primer, 2 μl (10 pmole) reverse primer, 16 μl DEPC-distilled (PCR grade) water and 5 μl DNA template were used. Non-template control, known ormation.P. falciparum and P. vivax DNA were made in order to mimic mixed infection in the following ratios: 100 μl:0 μl, 75 μl:25 μl, 50 μl:50 μl, 25 μl:75 μl, 0 μl:100 μl). The results from these ratios were compared with experimental samples. Thirty-four (20%) of the samples were replicated twice to validate reproducibility of the experiment.An artificial mix of Plasmodium falciparum infection was represented at 75°C to 78°C temperature range. Plasmodium vivax mono-infection was represented at peaks lied on 80°C or above. Plasmodium ovale was determined from the experimental set up and recorded at 79°C. This result was confirmed from published paper results which used the same primer pair and experimental set up[P. falciparum and P. vivax. In order to confirm both single and mixed infections the known positive controls of P. falciparum and P. vivax species were included in each experiment. The single or dual melting curve peaks and temperature profiles obtained from the positive controls were compared with melting curve peaks and temperature profiles obtained from the clinical samples  was selected from melting curve window. The shape and peaks of the melt curve was carefully observed. The dual and single melting curve peaks were observed from 73°C to 82°C. The most curve peaks were clustered from 77°C +/- 1. The melt curve peaks were shown as discrete variable values. If the curves were observed as dual sharp peaks, more than a difference of 1.5°C, then the peaks were recorded as mixed infections. If the curve was observed as a single sharp peak the result was recorded as mono-infection. l set up. If dualThe real-time PCR results were recorded from amplification curve and melting curve after each run of PCR. The amplification curve in threshold and log scale format was obtained from the Exicycler software in order to observe target gene amplification and the presence of primer dimers, non- specific amplification respectively. The amplification curve in log scale form clearly presents the exclusion of the “noise” or non -specific amplification and primer dimer in the left bottom side of the curve. The default auto-threshold and log scale were used to obtain these curves.P. falciparum or P. vivax single infection or a mixed infection of both species. The report did not include P. ovale and P. malariae infection. Among these positive cases 1,216 (67.48%) were infected with P. falciparum infection, 553 (30.68%) with P. vivax, and 33 (1.83%) with both species. According to microscopic diagnosis record, P. falciparum and P. vivax infection were the most common Plasmodium species with first and second prevalence rate, respectively, in the study area[Out of the 7,343 febrile patients who were attending the outpatient department for malaria diagnosis, 1,802 were reported to have either Among the 1,802 positive patients, 0.83% were below one year old infants, 6.71% were children between one and four years of age, 12.93% were between five to fourteen years of age, the remaining 79.52% were above fifteen years of age. This report showed that the different exposure rate of infections with regard to age group. Among 1,802 positive patients, 80.85% were males and 19.15% were females. Males were field workers and more infected than females which suggest a higher exposure to mosquito bites. Insecticide-treated bed nets are not commonly used by field workers in the study area, and this could explain the different rate of infection.P. falciparum mono-infection was considered in all study sites to compare its prevalence in each study site. The prevalence was higher in Metema Hospital than the rest of study sites. The hospital is found in relatively lower land than the other study sites. It provides medication for many people compared to other study sites. In addition, this hospital is found in an investment area in which seasonal labour workers come to the area where they are exposed to malaria infection  were P. falciparum, and three (1.79%) were mixed infection of both species microscopically. The re-evaluation of these 168 samples by SYBR Green I-based real-time PCR method and based on melting curve peaks indicated 112 (66.7%) P. falciparum, 10 (5.6%) P. vivax, 17 (10.12%) P. ovale, and 21 (12.5%) were P. vivax + P. falciparum mixed infections. Eight (4.8%) were negative for any one of the malaria parasite species. Plasmodium ovale was not commonly reported by microscopic diagnosis in the study area. Although P. ovale wallikeri and P. ovale curtisi have been reported by species-specific nested PCR in Dembia district, which is the same study area[P. vivax was not concordant with other studies. Other recent studies showed that P. vivax is second highest malaria species in malaria endemic area of Ethiopia[Out of the 168 samples 7 (4.17%) were udy area, they weEthiopia.The prevalence of malaria parasite infection obtained from Giemsa stain and its re-evaluation using the SYBR Green I-based real-time PCR method shows a high discrepancy Table . Real-tiIn this study, the detection process in both amplification and melting step was approximately 2 hours and 20 minutes and Plasmodium species were detected successfully. Melting curve analysis has an advantage to observe all Plasmodium species simultaneously in a single step. In addition, the method enables closed system data analysis in which it prevents post-PCR contamination of the samples unlike gel electrophoresis.. The misdiagnosis of single or mixed infections leads to mistreatment of patients with inappropriate drugs[The use of microscopy for febrile patient diagnosis does not have reliability for effective patient treatment. SYBR Green I-based real-time PCR targeted 18S rRNA gene is sensitive and specific to detect all the four types of Plasmodium species, 24. Thete drugs.The results indicated that malaria is a public health concern in the study area and there is misdiagnosis of parasite species as single and mixed infection. The discordant or mismatched results obtained from microscopic diagnosis and SYBR Green I-based real-time PCR amplification showed that clinical diagnosis of malaria species and detection of mixed species needs special attention in the study area. Even though real-time PCR based species diagnosis is expensive for clinical application in remote areas, the method is highly advantageous to control and manage prevalence of mixed infection, right representation of single species infection, and detection of emergency of drug resistance strains. Clinical diagnosis quality control strategy is important in malaria endemic areas for prompt treatment and control of the disease.


Intermittent mass screening and treatment (iMSaT) is currently being evaluated as a possible additional tool for malaria control and prevention in western Kenya. The literature identifying success and/or barriers to drug trial compliance and acceptability on malaria treatment and control interventions is considerable, especially as it relates to specific target groups, such as school-aged children and pregnant women, but there is a lack of such studies for mass screening and treatment and mass drug administration in the general population.A qualitative study was conducted to explore community perceptions of the iMSaT intervention, and specifically of testing and treatment in the absence of symptoms, before and after implementation in order to identify aspects of iMSaT that should be improved in future rounds. Two rounds of qualitative data collection were completed in six randomly selected study communities: a total of 36 focus group discussions (FGDs) with men, women, and opinion leaders, and 12 individual or small group interviews with community health workers. All interviews were conducted in the local dialect Dholuo, digitally recorded, and transcribed into English. English transcripts were imported into the qualitative software programme NVivo8 for content analysis.There were mixed opinions of the intervention. In the pre-implementation round, respondents were generally positive and willing to participate in the upcoming study. However, there were concerns about testing in the absence of symptoms including fear of covert HIV testing and issues around blood sampling. There were fewer concerns about treatment, mostly because of the simpler dosing regimen of the study drug (dihydroartemisinin–piperaquine) compared to the current first-line treatment (artemether–lumefantrine). After the first implementation round, there was a clear shift in perceptions with less common concerns overall, although some of the same issues around testing and general misconceptions about research remained.Although iMSaT was generally accepted throughout the community, proper sensitization activities—and arguably, a more long-term approach to community engagement—are necessary for dispelling fears, clarifying misconceptions, and educating communities on the consequences of asymptomatic malaria. In recent years, much progress has been made in the fight against malaria, resulting in substantial global reductions in mortality and incidence rates—a result of increased funding and commitment to prevention and treatment strategies , 2. HoweThere is increasing recognition of the complexities that come with implementing community wide health interventions like intermittent mass screening and treatment (iMSaT), mass drug administration (MDA), insecticide-treated bed net (ITN) distribution, indoor residual spraying (IRS), and intermittent preventive treatment (IPT), which are aimed at malaria control and prevention –7. OkellDuo-Cotecxin®, Holley-Cotec Pharmaceuticals Co. Ltd., Beijing), taken once a day for a total of 3 days. Nested within the iMSaT study is a qualitative data collection component to document community perceptions on the iMSaT intervention (including testing and treating) before and after the first implementation round. This paper will present the findings from this latter component.The Centers for Disease Control and Prevention (CDC) and the Kenya Medical Research Institute (KEMRI) have been collaborating on malaria research and public health programmes in western Kenya for over 35 years. Researchers are currently evaluating iMSaT as a possible additional tool for malaria control and prevention in Siaya County, western Kenya. The iMSaT study will assess the impact of repeated rounds of mass screening and treatment on malaria transmission, morbidity and mortality over a 2–3 year period. In this strategy, every individual within a community is screened for malaria with a rapid diagnostic test (RDT) at the household level, regardless of the presence or absence of symptoms, and persons found to be positive are treated with dihydroartemisinin-piperaquine  was introduced as first-line treatment for uncomplicated malaria and was provided in government and mission health facilities; in 2012, the Kenya Ministry of Health adopted a universal policy requiring diagnostic confirmation of all individuals with reported or documented fever where this is possible before they are given treatment for malaria in facilities where this is possible ? What do you think about it?P4: It’s good because it immediately gives out the result. That’s one of its advantages that I saw; it gives a genuine result. I think it’s good for our life as it gives results very fast whether there’s electricity or not. It will just give results of what you want tested. .Nevertheless, some who had never been tested with an RDT (and even for some who said they had) associated it with the HIV testing kit and thus believed the RDT could be used for diagnosing both malaria and HIV.  KEMRI-CDC is planning a study where they will visit people in their homesteads to test them for malaria even if they do not have any malaria symptoms…They will use a type of malaria test that provides results within 20 min [showing the RDT].M: P6: Yes I have seen this, isn’t it used for testing blood?P3: Yes… it’s used for testing blood…P4: Isn’t it used for the HIV test?M: This is only for malaria.P4: But it looks like the other one. When discussing whether the community would accept being tested with an RDT in the upcoming intervention, this association of the RDT with HIV testing led some community members to suspect that iMSaT staff might pretend to test for malaria but actually test for HIV.Many people will have different opinions especially now that we have many HIV cases, they will just say that the CDC are pretending but they are testing for HIV. Some will think that they are going to be tested for HIV instead of malaria. Additional concerns about testing centered on the issue of giving blood. Many respondents believed that too much blood is taken when they participate in clinical studies while others do not like having their finger pricked. Others expressed concerns about where the blood samples are taken and what they are used for.You know that when you come in the name of CDC, like even some of us whose children were taken by CDC, it’s what will make people refuse to participate in this study. When they hear of blood samples [being] taken by the CDC group, they will tell you that they take too much blood in a full syringe like that of a cow, but another person will say that a little blood sample is taken for test. That is what will make people refuse to participate in the study because CDC has a bad reputation. Majority say that they remove lots of blood from people and take it somewhere and when a child dies they remove the body parts. And as a result people get scared about CDC tests but I welcome it because I have found help from it. There was a wide range of understanding regarding the purpose and benefit of the iMSaT study. Although the concept of asymptomatic malaria infections and the purpose of treating them was explained during initial sensitization meetings, not all of those interviewed recognized the value of testing in the absence of malaria symptoms. This lack of understanding of asymptomatic malaria led to a variety of concerns with both testing and treatment.  M: In your opinion, how do you think people will feel about being tested for malaria with an RDT when they do not feel sick?From my experience I cannot accept to be tested when I don’t feel sick…I will wait until I feel sick to be tested.   P1:M: How do you think people will feel about malaria testing when they do not have any symptoms?P11: On this people’s perceptions will differ, some may send them away, those with knowledge who have heard of this will accept; therefore people’s perceptions will differ [depending] on how knowledgeable they are. There were some participants, however, who communicated an awareness of asymptomatic malaria—some more clearly than others—and thus recognized more readily the value in testing and the purpose of iMSaT. Respondents used a variety of phrases and ideas to convey this, such as the concept of “hidden malaria.”There [are times when] you may feel that you don’t have malaria, but you have hidden malaria, therefore it is only a test that can show whether you have malaria. Someone can live with malaria in their bodies for long without knowing it…you think you don’t have malaria but it’s already in you, so if you get an opportunity to be tested before the malaria symptoms show in your body then that will be a good opportunity. Compared to concerns about testing in the absence of symptoms, there was less concern expressed about receiving treatment in the absence of symptoms (once tested). Most participants felt that if the test showed they were positive for malaria, they would welcome the treatment without hesitation. When participants were asked about their perception of the study drug Duo-Cotecxin, some drew on their previous experiences with the drug which had been introduced fairly recently in some clinics but was not yet widely accessible. The majority of responses about Duo-Cotecxin were very positive—mostly due to its perceived high effectiveness and simpler dosing regimen as compared with the current first-line drug Coartem, which people perceived as having lost its “power” and being quite burdensome in terms of dosage.It will be easy for them for now…Coartem and the other one, even when taken for days, it’s not effective. You take and after 1 week or 2 weeks the malaria is still the same. It is less, so you find that with [Coartem], one gets tired with taking it on the way and abandons it because you take four tablets and two tablets of Panadol, so it’s not easy…we can encourage them that this new drug is easier. This discussion of adherence issues emerged as a central theme as many participants highlighted the importance of completing the full treatment dose although admitted they often fail to do so.Depending on the doctor’s prescription, even when feeling well, it is better for a patient to continue taking or finishing the dosage. That is why it is not possible to cross a river without passing through water. I notice some change then I know the medication I was given has helped me, but if I don’t notice any change it could be that I got lazy and did not finish my dose and so the sickness persists, so I just take it for granted that the medicine was not effective but I am the one who did not complete the dosage. Other reasons for not completing the full dose may be due to adverse side effects or wanting to save partial doses for when they are ill again.In our community people like dividing drugs to others, somebody takes half and the other people also take half dose. Therefore with us we may see that the drug is efficient, so if somebody is given drugs, he should not share until the dose is over so that habit should stop. Although the majority of comments surrounding Duo-Cotecxin were positive, some participants cited ‘fear of new things’ as a potential reason for refusal to take the drug.  M: What are the possible reasons for refusals?P2: Some people fear…such that anytime new things are done they are just suspicious. That could be a reason for refusal…P4: …you do not want to change your mind, ideas of the past, that’s why people fearP5: People always fear new things. For some people think…‘These new things, do they want to spoil us or do they want to make us good’? .An additional concern included the potential side effects of Duo-Cotecxin, but some participants suggested that most community members would comply with the treatment as long as it proved effective and did not cause too many side effects.Participants also wondered about the affordability and availability of Duo-Cotecxin based on their previous experience with the high cost and/or limited drug supplies.The concern they may have is, in case the price can go down, that it be locally available, not only at [name of local private health center]. It should be looked into that the pill becomes available in government hospitals so as to reach the common man. Participants were given the opportunity to provide suggestions on what should be done to encourage people to participate in the study and to address concerns with testing and treatment. There were many recommendations for further sensitization in the communities in order to raise awareness and clarify any misconceptions about the purpose of the study. While some participants recommended individual counseling during the screening and treatment process, others felt that schools and churches were the ideal pathways for sharing information.It depends on someone’s understanding. It is a good idea for a person who is doing the testing to talk or counsel the person tested for malaria to understand the results that show he is positive for malaria but still they do not feel sick. What we think is this: if you want to come for such occasions, tell us in advance. Just go to the school…when it is announced in schools, our children are there, and they will then tell us that the malaria team will come tomorrow. It is easily done through schools. We get information from schools through teachers…the only way to help us is to send messages through the teacher…or through the church, the church on Sunday. Many participants, especially opinion leaders, believed they could also play a role in sensitization by talking with community members and sharing the information they had learned during the focus groups.We should encourage each other by involving one or two people who have been trained here in [village] to educate more people…on what the exercise of mass screening is going to entail. Then educating more people on what the testing and treatment of malaria is all about. After the completion of the pre-implementation round, preliminary results with respect to community suggestions about the need for sensitization were promptly shared with the wider iMSaT study team. Prior to implementation of iMSaT, a round of sensitization activities was conducted in study villages targeting the local community, opinion leaders, community advisory board (CAB) members and CHWs with the aim of addressing study concerns. The iMSaT community sensitization team visited communities to explain how the study fits within the agenda of malaria control, introduced thematic areas of the study , and discussed basic procedures of participation/selection and potential benefits of participation.Findings from the qualitative interviews conducted after the first round of iMSaT was completed indicate that many participants were positive about their overall experiences during the iMSaT household visits and expressed their appreciation and willingness to continue with the study. Compared to the pre-iMSaT round, the comments during post-iMSaT interviews also tended to be more positive about KEMRI-CDC.  M: What are people in this community saying about the iMSaT study?P3: We as those people who were tested are really positiveP8: After doing the rounds, people from this community appreciated the activities of the iMSaT studyP5: I have heard most people talking positive about the IMSAT activities.   P5: What I hear people saying is that the study has really helped people. Even those who didn’t feel sick were found with malaria and were given drugs. So they appreciated that the study was goodP1: That’s what I heard…people appreciating, and I also appreciated it. Some respondents emphasized the importance of the study team’s attitude and their exchange with community members during household visits. They also highlighted the perseverance of the iMSaT teams as they returned to households in attempts to screen missed groups.The team worked very well. In fact, there are places that they could go to and find children playing outside; they would play with those children first to calm them down owing to the fact that children are scared of being injected/pricked…so they would play with those kids, soothe them until they accepted. The team worked very well. They were humble and walked in peace. P4: They were very humble people…aah! Because when they came to my house, I was in the garden but they took their time…then they tested me, they were so humble and patient people.P2: I loved them because when they came, they could teach first what they were to do and only when you agreed with their programme is when they would go ahead with their testing and treatment. Despite the overall positive comments about the iMSaT intervention, concerns about malaria testing remained. Many participants still expressed concerns about being tested for HIV or had heard rumours that the study teams were using malaria as a ploy and actually screening for HIV.The challenge I encountered though small was that some people would accept to be tested and others wouldn’t. The ones who would refuse would ask questions such as ‘this test you’re doing isn’t for malaria. You’re lying to us that this test you’re doing is for malaria, isn’t it?’ Most people in this community fear the tests as they always think that every test being conducted is for HIV/AIDS…so that’s why you see people running away because they fear the tests. The issues around blood samples—both fear of giving blood and confusion as to where the samples were taken—again proved to be a main source of unease or dissatisfaction with the process, and oftentimes, a reason for refusal to participate in the study.Some people feared that they have little blood and [said] the iMSaT teams were like blood suckers. The reason some refused was that most people, even those who live in this village, think the KEMRI people want to sell their blood. During household visits, a finger prick blood sample was also collected for preparation of dried blood spots on filter paper for assessment of RDT sensitivity. Respondents were particularly confused about this use of filter paper in addition to RDTs. It is unclear whether this confusion was due to their own misunderstanding or rather to lack of or misinformation given by the iMSaT teams.After testing me, my blood sample was taken on the piece of paper for further screening. It’s not easy for me to get the result…this is not easy for us to understand…why the result cannot be given back to me, to know what is going on or what was found wrong in me. I can remember being told that it would be taken to Atlanta, somewhere in Atlanta but the details I’ve forgotten when they said it would be back but remember being told it would be taken to Atlanta somewhere. As anticipated from the pre-implementation round data, there were fewer concerns with the treatment component of iMSaT, and it was rarely cited as a reason for refusal. Many participants expressed their appreciation for the treatment and praised the drug’s effectiveness.Duokoteko’ meaning ‘giving energy’ in the local language.It was discovered that some community members  had adapted the name of the study drug from Duo-Cotecxin to ‘What I saw interesting during the visit was…when a person who was screened and found to be malaria positive, then Duo-Cotecxin was given, and she got healed and felt much better so from that time she started calling Duo-Cotecxin drug ‘Duokoteko’ [laughter]. No, we have no problem because it is the same as…it’s like you are saying some Luo word…‘Duokoteko,’ therefore you know it’s like it reduces pain [laughter]. M: And what’s that, ‘Duokoteko’?P2: There are some people who had malaria…and after being tested they were given drugs which made them have more energy, that’s why they called it ‘Duokoteko.’ There were some participants, however, who believed they were being used as “guinea pigs” to test the effectiveness of Duo-Cotecxin.It’s like we are being used as guinea pigs to conduct study trials with, yet there is nothing that we are given. Personally, I am not happy with the study activities because we are not gaining anything at all. Why did these people refuse to participate in this study?M: P2: Some people tend to think too far when help comes. They think that the CDC people conduct research on them when they discover a new drug abroad then they come to test it on them to find out if it’s effective. So the community members say they are not monkeys for research to be conducted on them…some people touch on that first then they wait to hear from others about the effects. A common sentiment among those who tested negative was disappointment they had not received any drugs.My perception was, I thought after screening I could be given some medicine, even if I was not malaria positive. I could be given some medicine for emergency in case I fall sick but I did not see [it]. Conversely, among those who tested positive and were given treatment, it seems that some may have failed to finish the full treatment dose.  M: I would also like to ask you a question, with those who took the medicine, how were they…and even today?P2: I would say that most of those people did not finish the dose. P4: I have never been at ease with these new drugs because they could have adverse effects that come laterM: So, haven’t you taken the drugs you were given?P4: I haven’t taken them [laughter]. They do have adverse effects… it’s something still under trial. Those praising the drug may come to cry later. Respondents again encouraged further sensitization as well as follow-up visits so as to improve participation in subsequent iMSaT rounds. Many respondents commented on the poor sensitization leading up to the study, or lack thereof, and believed it to be the main source of participant refusals.This was due to the fact that they were never sensitized…Because all this work was left to the CHWs, and no village elders were selected to help in the mobilization on the impending visit that was to come…So you find that the CHWs come with some bag and explain to you what is going to happen, and since proper sensitization was not done, some people stayed away. I would respond by saying that some people refused because of lack of knowledge. Some people would refuse because they don’t know the benefits of what has been brought to them and its goodness. So my advice is before [iMSaT] round 2 begins, mobilization and sensitization should be done in the community. The community members should be able to speak for themselves and say that when the CDC people brought us treatment for malaria, I felt this way after taking the medicine. Some noted that with more sensitization, people would change their mind about participating in the study after seeing positive results from the first round of treatment. However, it is important to note that there were refusals even among those who received sensitization before the study.  M: For the people who refused…didn’t you carry out sensitization?P2: Sensitization was carried out, but it is difficult for people to understand things. Some people would accept and even sign the consent form. Just before you begin your work they say, ‘No, test these children and leave me. She is cheating me and what I’m seeing here resembles the HIV test kits. You want to test for HIV, so just test the children and leave me alone.’ So there’s nothing you can do- you just test the children and leave her alone. Data from the iMSaT study indicated that refusal rates were relatively low, although the number of refusals nearly doubled in Round 2 of iMSaT. A 3.3 % refusal rate was recorded in the first round of iMSaT with a total of 23,199 people sampled and 755 refusals. Round 2 and Round 3 of iMSaT  resulted in refusal rates of 6.0 %  and 5.6 %  respectively.Overall, there was little relevant variation across and within groups: although female respondents seemed to express slightly more concern regarding testing and treatment as compared to their male counterparts, they also tended to share more positive statements about the study . As might be expected given their role within the community, opinion leaders tended to be more outspoken and more readily expressed criticism of KEMRI-CDC and research as compared to other respondent groups. When CHWs were asked to discuss community perceptions and opinions around testing and treatment, their comments tended to align with those of the community members themselves.Qualitative interviews with community members and opinion leaders revealed mixed opinions of iMSaT activities. In the pre-implementation round, respondents were generally positive and willing to participate in the upcoming iMSaT study. There were, however, some concerns related to testing in the absence of symptoms including fear of being tested for HIV and issues around blood samples. There were fewer concerns about the study drug Duo-Cotecxin although these included affordability and future availability, but most respondents were positive about the treatment due to its simpler dosing regimen. The pre-implementation data were primarily based on hypothetical scenarios or experiences outside the iMSaT study (in order to foresee any major problems with the intervention); however, these issues were largely confirmed in the post-implementation data. After the first round of iMSaT, there were less concerns and negative views expressed, although some of the same issues around testing remained, as these were the most common reasons cited for refusal. Although not the primary focus of this study, community perceptions of the KEMRI-CDC research and public health collaboration cannot be divorced from the perceptions of the iMSaT study. The results suggest that participants’ views and acceptance of iMSaT activities were heavily influenced by their perception of research as a whole and their previous experiences with other studies.The majority of concerns during both pre- and post-iMSaT centered on fear of covert HIV testing and issues related to having samples of blood taken . These findings are consistent with both Silumbe et al.  and OkelRumours about blood  are quite common and widespread across sub-Saharan Africa , 21, 22 Among community members, it was clear that knowledge of malaria was high, as was their awareness of the burden and consequences, but there was less awareness of the important role of asymptomatic malaria, which may have hindered full understanding and acceptance of the study. These findings are again consistent with Okello’s study , which cThe iMSaT findings highlight the importance of educating participants about adherence and suggest that follow-up visits should continue to be an important and necessary component of iMSaT in order to ensure proper adherence to the study drug (as some respondents suggested in their recommendations). However, some of the same iMSaT participants who admitted to taking partial doses of the study drug also emphasized the importance of completing treatment as prescribed and conveyed an understanding of the dangers of not finishing the full dose. This discordance between knowledge and behaviour suggests other underlying external factors may be at play (outside the iMSaT study) such as access to treatment, cost or availability of drugs, poverty, and other structural issues.Another finding worth highlighting is the study teams’ attitude and perseverance which participants remarked upon throughout the study. Studies of MDA in the neglected tropical diseases  have found that the type of drug distributor  and how and when they distribute are often key factors in the acceptability and uptake of MDA , 26. ThiThe themes which emerged during the pre-iMSaT round of qualitative data collection appear to have anticipated the barriers and challenges for household visits and highlight the need for further, targeted sensitization activities. The sensitization activities that took place may not have been as comprehensive or as effective as expected. Given the number of comments on poor sensitization (or lack thereof), and the emphasis participants’ placed on issues of blood and HIV, it is likely that some refusals or withdrawals were largely due to misinformation or misunderstanding of the purpose or intentions of iMSaT (as discussed above), or of medical research as a whole—concerns and beliefs which may have existed for many years already. Addressing these will prove challenging; fears related to blood are not easily dispelled and may actually be linked to deeper issues of power imbalance and social distance between researchers and communities . Dial’s Given the lack of detailed documentation with regard to how the iMSaT sensitization activities were conducted, it is difficult to know whether or not participants’ concerns were adequately addressed before the first round of iMSaT. Regardless, it is also important to recognize that these issues may not be merely a question of health education, but may require a more long-term, culturally appropriate approach to community engagement and researcher-community dialogue.For this purpose, KEMRI-CDC has in place a community engagement strategy, which is overseen by a Community Liaison Officer. Activities include informing the community of the objectives of planned and ongoing activities, generating community acceptance and support for the programme, community mobilization for informed participation, promoting a positive image of the organization, coordinating result disseminations to community members, coordinating outreach activities, and managing rumours and propaganda arising from research activities. There is a rich set of literature exploring these elements of community engagement and offering insights from programmes which have attempted to put them into practice in various contexts –31. AlthThe qualitative study achieved its original objectives of investigating pre- and post-implementation perceptions of screening and treatment in the absence of symptoms, and the results served to inform delivery of the iMSaT intervention. The iMSaT study (and future KEMRI-CDC research) would likely benefit from more robust, better documented community engagement activities— specifically tailored to individual studies and to the concerns of participating communities. For the remainder of the iMSaT intervention rounds, however, a short-term variation of this approach could be considered in order to open communication channels and further clarify misconceptions, thereby improving the acceptability of the study and ensuring optimum uptake and adherence to the intervention. Although community acceptance of iMSaT was generally positive, sensitization still needs to be refined and adapted to specific community concerns and misconceptions as outlined in this paper. It is imperative to have accurate, consistent, and frequent communication from researchers to community members and vice versa.Following suggestions from community members, it is clear that future sensitization should continue to incorporate village leaders and other trusted group leaders (youth or women) who play an important role in community buy-in, as discussed in , 27. TheThe findings from this study have also raised some important questions regarding the issue of adherence and to what extent it is influenced by sensitization efforts. The data touched on perspectives from iMSaT participants who appear to understand the consequences of not adhering to treatment and yet choose to not finish their dose for various reasons. The community members who are continuously sensitized and educated yet fail to follow through illustrate the frustrating complexities of community health interventions and drug compliance. A more realistic approach may be to accept that there will always be a proportion of people who will not benefit from sensitization and may not adhere to treatment. Some may argue there is a need for further studies on this issue—one which has extensive implications for the success and impact of future public health interventions.Most of the pre-implementation data were based on hypothetical scenarios of testing and treatment and thus may not have identified the full range of factors influencing participation that could occur in later rounds. They may overestimate or overemphasize the importance of negative perceptions which appear to have had little effect on actual participation given the low refusal rates in the first three rounds of iMSaT. Only six out of a total 41 intervention communities were included in the qualitative component. However, the number of interviews conducted (36 FGDs and 12 CHW interviews) comprise a fairly large sample of respondents for a qualitative study, and the data suggest that a level of data saturation was achieved as no new themes emerged in the final interviews. In addition, despite random selection of FGD participants, not all community members on the original lists were available at the time of interview. Consequently, village leaders sometimes resorted to convenience sampling, appointing available community members as replacements. Nevertheless, the study benefited from triangulation of information from several types of participants: CHWs, opinion leaders, and male and female community members of varying age, as well as from all three districts in the study area. Although data quality control strategies were implemented in an attempt to minimize potential bias from the transcription process, there are factors which might have influenced transcript content. These include translating Dholuo recordings directly into English (bypassing written Dholuo), which might have resulted in variations in facilitators’ interpretations of both local language nuances. Although the small number of communities included in this study may limit the extent to which the results are generalizable to other contexts, the findings are consistent with other studies that have examined community perceptions of research trials, in Kenya and elsewhere.This qualitative study has explored community perspectives of intermittent mass screening and treatment for malaria and has identified barriers to acceptability and implementation. The findings have implications for future mass treatment interventions as well as for wider efforts in community engagement. Further sensitization activities may help alleviate participants’ concerns by dispelling fears, clarifying misconceptions, and educating communities on the role and consequences of asymptomatic malaria (as shown in other studies). It is necessary, however, to approach community engagement and sensitization activities with realistic expectations; there will likely be a portion of community members who are not very receptive to one-off sensitization activities. A more long-term commitment to community engagement and communication may improve relations and foster trust between such community members and those conducting the research. Researchers and programmers should re-focus their attention on the growing body of information on community engagement in practice in order to build upon existing models of how research institutions and programmes can best integrate their work into study communities—thereby facilitating acceptable yet rigorous research which is more likely to have a lasting impact on the populations it serves.


Plasmodium vivax incidence in Henan Province. As China is in a pre-elimination phase, the surveillance of imported malaria is essential, but there is no good way to distinguish imported cases from indigenous cases. This paper reports a case of a 39-year-old man who acquired P. vivax while staying in Indonesia for one month in 2013, and relapsed in Henan, China in 2014. This was diagnosed as vivax malaria based on rapid diagnostic test, Giemsa-stained peripheral blood smear and Plasmodium species-specific nested PCR. The genetic sequence for the circumsporozoite protein genes was analysed and the genetic variations were compared with a previously constructed database of Chinese isolates. The results from the circumsporozoite protein (CSP) gene sequence analysis centered on the repeat patterns showed that the imported cases had completely different sequences from any subtypes from Chinese isolates, but well matched with the countries travelled by the patient. The imported vivax cases were able to clearly distinguish from the indigenous vivax cases by detecting the CSP gene and were able to confim its origin by genotyping.In recent years, there has been a substantial increase of imported  Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Globally, between 135 and 287 million people were infected in 2012, of which over 600,000 died[Malaria is a parasitic infection caused by Plasmodium vivax was endemic in the whole province, while P. falciparum was prevalent in the regions south of 33° latitude north. In the early 1970s, the morbidity reported was 16.9%, the number of the malaria cases was highest in the country[In Henan Province, malaria has been a major health problem historically.  country. Afterwa country. TherefoP. vivax from Southeast Asia, who relapsed three months after leaving there, this case was diagnosed by light microscopy, rapid diagnostic test(RDT), nested PCR and sequence analysis. It was the first imported relapse case recorded in Henan Province.This study aimed at a case of imported During the work of anti-relapse treatment in 2014, a malaria patient was met by chance, who had a malaria history last year. The patient was a 39-year-old Chinese man with a one-day history of fever (38.1°C), shaking chills and headache; he reported no other constitutional symptoms and without underlying diseases.nd December 2013, he suffered a sudden onset of rigors. He was diagnosed with malaria on 6th December 2013. This case was microscopically identified to be P. vivax  for malarial antigen was positive. The peripheral blood sample showed  Figure . The denThe patient was treated with dihydroartemisinin and piperaquine phosphate tablets  and primaquine (22.5 mg/day for 8 days). He had a rapid and favourable response, his symptoms began to subside after one day of treatment, on the 10th day, the parasites were not observed.Plasmodium genus-specific set (rPLU1/rPLU5) was used for the first-round PCR. Other four pairs of species-specific primers  that had been reported in a previous study were used in the second-round PCR[P. falciparum, P. vivax, P. malariae, P. ovale) and pure water was used as the positive and negative controls, respectively. The amplified DNA product corresponded to P. vivax (120 bp). The presence of P. vivax in the patient’s specimen was confirmed by nested PCR.For the 18S rRNA, nested PCR was performed. DNA was extracted from the patient’s blood samples with the QIAamp DNA Mini kit  according to the manufacturer’s instructions and stored at -20°C. ound PCR. Nested et al.[The polymorphic regions of the circumsporozoite protein (CSP) genes from the DNA samples were amplified and sequenced. The primers and amplification conditions were described by Imwong M et al.. The CSPet al., one seqMultiple sequence alignment of all the genes sequences and loci was performed with the algorithm Clustal X software. Phylogenetic trees were constructed using the Molecular Evolutionary Genetics Analysis(MEGA) 4.0 and their reliability was tested by bootstrapping analysis . One cluster was considered significant if it was present in more than 75% of the permuted trees Figure .Figure 2P. vivax infection, had fever, chills, headache for one day, but no other constitutional symptoms. Meanwhile, patient’s current residence, Lankao is a non-endemic area for malaria. Only considering his travel history and historical exposure to P. vivax, given the related examination of malaria, the result was positive. This case suggested it was necessary to obtain the epidemiological history when formulating a diagnosis and it also highlighted the importance of considering more etiologies for febrile illnesses, which was especially important in the current era of globalization and increasing residential mobility[The case with a history of mobility.P. vivax case from an endemic area in Henan Province. Malaria relapse refers to the re-emergence of the malarial paroxysm after several weeks to more than one year without malarial parasites infection when the parasitemia in the erythrocytic stage has been eliminated at the first onset of malarial patients, which is possiblly related to activated hypnozoites. Relapsable malaria includes vivax and ovale malaria and other monkey malaria. This case reminded everyone, when facing a patient with fever of unknown origin, obtaining a thorough long-term residential and the history of the spread of malaria is very essential.This was the first report of a relapse nd December 2013, he was diagnosed with vivax malaria on the fifth day, the densities of malarial parasites were 330/μl at that time. Then he suffered a relapse on 23th March, was diagnosed on the second day, but the densities of malarial parasites were 5760/μl. The case also showed us, the patients maybe had more tolerance to the malarial parasites when they were suffering a relapse. So in the relapse patients, the disease would develop more rapidly than those with primary infection.The patient’s primary attack of malaria began on 2Plasmodium spp[P. vivax VK210 strain has CSP amino sequence that includes a GDRAA/DGQPA repeat[P. vivax is endemic.CSP is found in all the mature malaria parasites, which forms a dense coat on the sporozoite’s surface. It compdium spp. The P. A repeat, 13. A vA repeat. This poAccording to the previous studies, the amino acid sequence of this case in the CSP regions belonged to the VK210 type, 16. ForP. vivax during his stay in Indonesia rather than in China. Therefore, careful history taking, including the current residency and travel history, is important to make the diagnosis of malaria. Genotyping is a useful tool to determine the origin of vivax malaria and discriminate imported cases from autochthonous cases. It is very significant in the elimination era.In this particular case, the patient had been in Indonesia for one month about half year before and the clinical symptoms were not specific, however, malarial parasites were observed in the peripheral blood smear, the nested PCR also supported the result. Considering the test results and his travel history, it could be guessed that he was infected with Written informed consent was obtained from the patient for the publication of this report and any accompanying images.


Over-diagnosis of malaria among African children results in mismanagement of non-malaria infections. Limited laboratory capacity makes it difficult to implement policies that recommend pre-treatment confirmation of infections so a new approach with a package for on-the-spot management of fevers was evaluated.Febrile children presenting to outpatient clinic were randomized to receive either a ‘test-treat’ package  or routine outpatient care in a secondary health care facility in Kumasi, Ghana. A diagnosis of malaria, bacterial, viral or mixed malarial and bacterial infections was made using pre-defined criteria. Outcome was resolution of all symptoms including fever on day 7.vs 46.2%, p = 0.190) and mixed malaria and bacterial infections  but more diagnoses of viral  and bacterial infections only . Less anti-malarials  and antibiotics , were prescribed in the test-treat group on completion of study, more test-treat package patients were clinically well  and febrile  and less were admitted for inpatient care (0.0% vs 8.4% p = 0.001) compared to the routine care group.The median age of the patients was 37.5 months (IQR: 19 to 66 months), with 56.7% being males. Compared to routine care the test-treat package resulted in less diagnoses of malaria, (37.2% Test-treat package improves the effectiveness of outpatient diagnosis and treatment of children with fever and reduces inappropriate prescribing of anti-malarials and antibiotics. The package provides clinicians with the option for immediate diagnosis and treatment of non-malaria fevers. The test-treat package now needs to be evaluated in other settings including primary health care facilities. The treatment of febrile children makes up approximately two thirds of the paediatric outpatient workload in African hospitals . MalariaAppropriate prescribing or withholding of drugs for infections is not only important for effective treatment and to avoid unnecessary side effects but it is also essential to minimize the risk of organisms developing drug resistance. Although many hospitals have access to microscopy for the diagnosis of malaria , fever iPlasmodium falciparum malaria provide accurate diagnosis within a few minutes [To be able to rationally prescribe or withhold anti-infective agents clinicians need to have access to diagnostic tests for the most common infections and to use the test results appropriately as guide. Trials have demonstrated the effectiveness of ‘point-of-care’ tests such as malaria Rapid Diagnostic Tests (RDT) and urine dipstick tests for providing specific diagnoses of infections , 11 in t minutes –16 and a minutes . Urine d minutes , 17. In The study was conducted at the outpatient clinic of Suntreso Government Hospital (SGH), a secondary health care facility in Kumasi, Ghana between August and October 2009 during the high malaria transmission season. SGH is a district hospital with 57 inpatient beds. In 2007, 22,322 children were seen and treated at the SGH outpatient clinic and 55.7% were treated as malaria. The hospital laboratory performed blood films for malarial parasites, haemoglobin estimations, automated blood cell counts, urinalysis and stool microscopy, but had no microbiological culture facilities, and clinicians did not have access to RDTS.All children between 6 months and 12 years of age presenting to the out-patient department with a temperature above 37.5°C or a history of fever were recruited into the study after their caregivers have signed informed consent form. Children requiring admission or referral due to severe illness at presentation as defined by World Health Organization’s (WHO) Integrated Management of Childhood Illness (IMCI) criteria  were excThe test trest-package consisted of rapid diagnostic test for malaria, urine dipstick test for urinary tract infection (UTI) and examination of ear, nose and throat, chest and treat with antipyretic with or without anti-malarials and/or antibiotic for any patient presenting with fever or history of fever. This package assumes that urine dipstick test for UTI is comparable to urinalysis, and rapid diagnostic test is comparable to blood film for malaria parasites.Routine care comprised of outpatient registration, triage of patients by nurses, and care given by attending doctors to patient which consists of history, general examination, followed by either laboratory request or direct treatment based on history and examination by doctors at SGH. The test treat package (intervention) was tested against routine out patient management of fever by doctors at SGH (control). Patients randomized to the test-treat group underwent a structured clinical examination consisting of ear, nose, throat and joint inspection and chest auscultation after their history had been taken. A bed side malaria RDT for all Plasmodium species  was performed according to the manufacturer’s specifications. Thick and thin blood films for malaria was done for all participants in the intervention group. All the RDTs were done and interpreted by one of the authors (EA) to eliminate inter-observer bias. A urine sample was obtained from each child in the intervention group, either spontaneously or by catheterization for children under two years old who could not produce urine on demand. These were then immediately tested at the bedside for nitrites and leucocytes using combi-10 urine dipsticks. Samples that tested positive for nitrite and/or leucocytes were transported immediately to the microbiology laboratory of the local Teaching Hospital (KATH) for culture and sensitivity.Diagnoses made as a result of these clinical and laboratory examinations were based on predefined criteria Table . PatientPatients in the routine care group received the routine care provided by the clinicians at SGH. These clinicians manage patients based on the recommendation of the Ghana Health Service treatment guidelines . DecisioBlood and urine samples collected from patients in the intervention group were transported to a laboratory at KATH. The blood film slides were examined independently by two experienced microscopists for the presence of malaria parasites. Discrepant results were reviewed by a third microscopist who determined the final result. The purpose of comparing RDT and microscopy results was to determine the accuracy of the malaria RDT in the study setting. Urine samples were cultured to determine the specific pathogenic organisms and the antimicrobial sensitivity. The management of the children were based on the results of malaria RDT and urine dipstick test as well as clinical examination after history.Patients from both groups who received anti-malarial(s) and/or antibiotic(s) were requested to return for review on the second and the seventh day after recruitment. However, all patients, in the intervention group, who received paracetamol only were given appointments for daily follow up as the sensitivity of the RDT used in the study was less than 100%. Sensitivity of the RDT used in this study was 98.0% and specificity is 83.3%. Patients in both groups who failed to attend their follow up appointments were visited at their homes to ensure they were clinically safe. At these reviews all children underwent a clinical assessment and the result documented. The patients were assessed by the same sets of clinicians who had conducted the initial assessment and management in the respective groups.The primary end point for the study was an asymptomatic and afebrile child as reported by the caregiver/child on day 7. Secondary end points were no symptoms and no fever on day 2. Information was collected about the presence of fever and/or symptoms on days 2 and 7. Patients who had fever at the end of the seventh day were further assessed and managed appropriately. They were followed till recovery. All data were collected on a pre-designed form and entered in a Microsoft Access® 2007 database.Pre-trial power calculations indicated that 120 patients would be needed in each of the intervention and routine care groups to detect a difference of 15% in the absolute treatment failure rates between the two groups. In this sample size there was an estimation of a two sided type I error of 0.05, a power of 80%, an absolute failure rate of 25% in the routine treatment group and an anticipated non-response/dropout rate of 15% were used. The data were transferred from Microsoft Access 2007 to Stata 10.1  from which univariate analysis with point estimates and 95% confidence intervals of baseline characteristics of the children were calculated. Bivariate analysis was performed using the Fischer’s test. Based on blood film analysis, sensitivity, specificity, positive predictive value and negative predictive value for the RDT compared to malaria parasite blood film microscopy (gold standard) in this setting were also calculated.The median age of the patients in the study was 37.5 months (IQR: 19 to 66 months) with 56.7% being males. The median age, age range and sex distribution were similar in both groups. One patient was inadvertently recruited into the intervention group resulting in 121 patients in intervention group and 119 in the routine care group  patients in the intervention group. Only one patient in the routine care group had urinalysis performed and it was negative.There were significant differences among diagnosis between the groups in diagnosis at presentation Table . SimilarSeven of the 240 patients, three from the intervention group, failed to come for follow up appointments and they were traced to their homes and reviewed. This was to ensure that each patient was clinically safe. At day 2 review, only two (1.6%) of the patients in the intervention group required another laboratory investigation because of persistent fever compared to 13 (10.9%) in the routine care group (p = 0.001). There were significant differences in the clinical outcomes of children in the two groups by day 7. Fever had resolved in 120 (99.2%) of children out of 121 in the intervention group compared to 96 (80.7%) out of 119 in the routine treatment group (p = 0.001). No child in the intervention group required hospitalization whereas in the routine care group 10 (8.4%) children were hospitalized (p = 0.0001). One patient (0.8%) in the intervention group remained febrile at the end of the study compared to 12 (10.1%) in the control group (p = 0.001).In the intervention group 60  patients had positive malaria RDTs compared to 49  who had positive blood films by microscopy. Using microscopy as the gold standard, the RDT had a sensitivity of 98.0% (95% CI: 89.1% to 99.9%), a specificity of 83.3% (95% CI: 72.7% to 91.1%), a negative predictive value of 98.4% (95% CI: 91.2% to 100.0%) and a positive predictive value of 80.0% (95%CI: 67.7% to 89.2%).This study assessed the introduction of a simple test-treat package for management of stable febrile children in an African out-patient clinic in a secondary health care facility. The ‘test’ component of the package consisted of a structured examination of ears, nose and throat, joints and chest, a malaria RDT and urine dipstick testing. The ‘treat’ component was guided by the results of clinical examination and tests, and consisted of a choice of an anti-malarial (artesunate-amodiaquine), an antibiotic (co-amoxiclav) or an antipyretic (paracetamol). These drugs were used singly or in combination according to the diagnosis. The test treat package (intervention) was tested against the routine management (control) of stable febrile illness among children at the OPD by the doctors at SGH. The test-treat package resulted in resolution of fever in >99% of patients except one patient who remained febrile at the end of the study but no child in the test-treat package group required hospital admission.In contrast, 10.1% of the patients in the routine care had fever at the end of the study, and 8.4% required hospital admission. All patients who had fever at the end of the study were followed up till resolution. Almost all the patients in the routine care group who were prescribed anti-malarials had no laboratory confirmation of malaria infection although blood film microscopy for malaria was available in the hospital. International and local guidelines recommend that malaria should be confirmed if tests are available , 22 as tDespite policies promoting the use of RDTs to investigate fever there is still widespread over-prescribing of anti-malarials in the face of a negative malaria test. One reason may be that health workers often do not have any diagnostic tools for determining the cause of fever and, because they are concerned about missing a possible malaria infection, they default to prescribing anti-malarials. The RDT, which is highly effective in diagnosing malaria as a cause of fever, was included as part of the test-treat package, but the package also incorporated alternative diagnostic and treatment strategies for malaria-negative febrile children. For example, substantial proportions of patients in the intervention group had positive urine dipstick test and were consequently treated with antibiotics and not with anti-malarials. Failure to seek a definitive cause for fever in children puts them at risk of long term complications of the undiagnosed and untreated infections. For example, persistent, untreated urinary tract infections can lead to renal scarring, hypertension and finally chronic renal failure. In addition to the direct cost of prolonged or alternative courses of treatment and hospitalizations, ineffective management of febrile children prolongs ill health and necessitates repeated visits to health facilities, both of which generate an economic and social burden which disproportionately affects families , 23.The rate of anti-malarial use in patients who received routine care was 2.5 times higher than in children who were managed using the test-treat package. Globally, the successful control of malaria is dependent on the continued efficacy of artemisinins. There is already evidence that resistance to artemisinins is appearing in Southeast Asia , 25 and The study had some limitations. The two groups were managed by two different sets of clinicians. This resulted from the study design. The authors went to SGH to test their test treat package against the routine practice of management of fever by the clinician at SGH. It was also conducted during the high malaria transmission season and so the results may not be applicable to areas or seasons with lower transmission rates. However, malaria over diagnosis rates tend to be higher when transmission is lower  so our rThe potential benefits of such a simple and affordable approach for improving health outcomes in patients, for protecting the economic well-being of their families and for reducing the potential for drug resistance are immense.Ethical clearance was granted by the Committee on Human Research, Publications and Ethics of the Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.