However, empirical data from the field are still limited. The aim of this study was to assess whether training village volunteers on the use of Paracheck-Pf®) and an endline survey at six months follow-up. Survey data were supplemented by the analysis of logbooks and field-based verbal autopsies. Villages with midwives (MW) in post were used as a third comparison group in the endline survey. Intention-to-treat analysis was used.The study was designed as a cluster randomized controlled trial with a cross-sectional survey at baseline, a monthly visit for six months following the intervention  and 17 to the comparison group. The two groups had comparable baseline statistics. The blood tests provided by volunteers every month declined over time from 279 tests to 41 but not in MW group in 18 villages (from 326 to 180). In the endline survey, among interviewed subjects , the coverage of RDT was low in all groups  although the intervention  and MW  were more likely to receive a blood test. Mean (SE) of blood tests after onset of fever in days was delayed (intervention 3.6 (0.3); comparison 4.8 (1.3); MW 3.2 (0.4)). Malaria mortality rates per 100,000 populations in a year were not significantly different . None of the dead cases had consulted volunteers.The results show that implementing volunteer programmes to improve the coverage of accurate and timely diagnosis with RDT and early treatment may be beneficial but the timeliness of detection and sustainability must be improved. Plasmodium falciparum malaria, the World Health Organization (WHO) recommends early diagnosis by microscopy or with a rapid diagnostic test (RDT) prior to treatment [Late diagnosis and inappropriate treatment of malaria results in complications and mortality ,2. For ureatment ,4 with areatment . Using Rreatment -8. The sreatment ,9-12.Conclusive information on the use of trained community volunteers to expand ACT remains limited . FollowiP. falciparum and 40,167 to Plasmodium vivax, respectively and reports 972 malaria deaths [Myanmar is among 31 countries with the highest burden of malaria . Over 25a deaths . While oThe primary objective of this study was to determine whether the training of village volunteers and their regular supervision by health staff improve the coverage of timely diagnosis and treatment of malaria, to lower malaria-related mortality.Secondary objectives were (i) to compare the prevalence of malaria-induced fever in three groups of villages; (ii) to assess villagers’ knowledge of malaria transmission and of the free blood tests available; (iii) to compare trends in patients tested by volunteers and midwives; and, (iv) to determine RDT-positive rates for different age groups in selected areas.The study was conducted in Bago, a division in the south of Myanmar with 12,941 sq km of forested land and a population of 5,313,613 inhabitants. With an annual rainfall of 3,291 mm and temperatures ranging from 20.9°C and 32.3°C, malaria is endemic all year round with a peak in transmission in June and July. Six townships with moderate endemicity  were selected for this study . In 2008, 21 malaria deaths were reported by the six township hospitals but the number of deaths in patients who did not arrive at the hospitals is unknown. No volunteers had previously been trained in Bago Division.Farming and forest work are the most common occupations of villagers in the study area although seasonal work in plantations  or construction sites is frequent .Sketch maps were available in some health centres but villages were not indicated on the maps, and Global Position System (GPS) points were not available. In successive consultations with approximately 200 midwives, information for the study was collected including village maps, distance between villages and information about villages under their care. Villages with fewer than 70 households and those where midwives were stationed were excluded from the trial, as were villages presenting serious security problems. Only those remaining villages classified as “Stratum 1a” were included in the study . Thirty-eight villages at least two hours distance away from the nearest selected villages were then identified.The study population included villagers currently living in the endemic area or its surroundings for at least one month.A cluster randomized controlled trial, with each village as the cluster, was chosen because the volunteer was accessible to all villagers in any given village. Eligible villages (clusters) without existing health staff were then randomized into intervention and comparison groups. The nature of the intervention allowed neither allocation concealment nor blinding. Villages with midwives (MW) in post were used as a second comparison group in the endline survey , a RDT for the diagnosis of to 98.3% ,32. PatiA volunteer training module for malaria in Myanmar  and meals (17 USD) during their stay in Bago. Giveaways included a T- shirt, a cap and a handbag with a malaria volunteer logo.Following the training, a 16-item WHO RDT training evaluation instrument  was usedAfter completing the training, two boxes of Paracheck Pf® RDT (50 packets), AL and chloroquine were provided to all volunteers. Other materials included disposable gloves, a lancet, cotton, spirit, and a timer-watch, thermometer, calendar, patient record forms and a logbook. Monthly calendar cards were issued to record the availability of volunteers and their meetings with health staff. On returning to their villages, volunteers were to inform village leaders, seek their support in endorsing and promoting the programme, display two posters announcing the availability of free RDT, and report to local health staff for future supervision.Midwives in the nearest health facility routinely monitored and supervised volunteers during their immunization visits to villages (monthly). Malaria supervisors occasionally visited volunteers. The first author assessed the regularity of activities through monthly review of calendar cards and reports and regularly visited these townships to collect forms from malaria supervisors but did not supervise their activities.Data on village information and a listing of household members to screen for fever were collected. Subjects who had reported fever were interviewed by research assistants. Close relatives of the deceased were interviewed using the standard verbal autopsy guidelines . VolunteBackground characteristics, village population and malaria control activities were collected from village leaders.The baseline survey was conducted in the intervention and comparison villages between 8 March and 6 April, 2009. The endline survey was conducted in all villages visited for the baseline assessment, and in villages with midwives, between 21 November, 2009 and 17 February, 2010. Two teams consistently collected data for both baseline and endline surveys. They consisted of three research assistants from the Department of Medical Research - Lower Myanmar (DMR-LM) for the interviews, and a local health staff for the blood test.Two pre-tested forms  were used. The household form included village name, township name, a list of household members and their background characteristics such as age, sex, occupation, history of fever during the previous 30 days. The individual form included background characteristics, knowledge of malaria symptoms, number of febrile episodes, date of the episode, place of residence at the time of the episode, treatment type and place received, awareness of the availability and location where free RDT-based treatment was available, history of RDT and the main reason for use or non-use of RDT. The interval between the first symptom (fever) and blood test was assessed for timeliness of RDT use. Data in the endline survey were validated against information from volunteer logbooks.For each survey, all households were visited and data collected for the household forms. Villagers with high- or middle-school education completed the household forms under the supervision of a research assistant. All household members above the age of six months, living in the village for over 30 days and having contracted a fever within the previous month were eligible for interview. Those with fever for less than 24 hours were excluded, as they could not be assessed for timeliness of RDT. Appointments were made with eligible subjects for a face-to-face interview. When eligible subjects were under the age of 15, the mother or a close household member, were interviewed.During the baseline survey, village leaders were asked to keep a death register and ascertain the date of death in the endline survey. When reviewed however, the death registers were unavailable or incomplete. Dates of deaths were identified in several ways during the endline survey. First, the list of the deceased after 21 May, 2009 was collected from midwives, village leaders and the informal social group registers. Second, the close relatives of the deceased were requested to recall events on the day of the death of a kin. Third, the dates of death were confirmed with the death notices distributed locally. When a death notice was not available, recall was facilitated by the use of the Myanmar calendar, which includes a festival day every month.After identifying the deceased during the intervention period, the international standard verbal autopsy guidelines were used to identify causes of death in the post-intervention assessment, using questionnaires appropriate for each age groups . Two docTwo physicians, blinded to the intervention status, identified the causes of death from the verbal autopsy questionnaires. When a discrepancy between their conclusions arose, the final diagnosis was made by a senior doctor from the study area. All were independent from the control programme. Using the standard WHO case-definitions for malaria ,36, diagVolunteers’ and midwives’ (18) logbooks and patient record forms were reviewed from 21 May, 2009 until the end of the survey to determine trends in caseload and use of RDT.The primary outcome measures were: (i) whether villagers had received blood test-based treatment for fever, (ii) interval between onset of fever and the blood test, (iii) mortality. The secondary outcome variable was knowledge of malaria mode of transmission.The proportion of people anticipated to have received treatment within 24 hours after the intervention period was 50% in the comparison arm and 70% in the intervention arm, based on the 2005 Roll Back Malaria target of at least 60% of malaria cases receiving appropriate treatment within 24 hours of the onset of symptoms and the unavailability of information on proportion receiving malaria diagnosis within 24 hours from onset of fever information in Myanmar . The iniDescriptive statistics including percentages, mean and standard deviations were derived from volunteer background data, logbooks and patient record forms.The impact of the training was assessed using intention-to-treat analysis method, where two villages in the intervention group were included in the intervention arm although these villages did not receive training of volunteers. “R” software  and instThe surveys were analysed using sampling weights to compute estimates and standard errors for outcomes. Sampling weights were calculated using the inverse of the probability of selection, or the estimated total village population divided by the number of villagers screened for fever.The analysis of knowledge in interviewed subjects who had contracted fever used a scale from zero to eight for eight means of transmission, with a correct answer for each mode of malaria transmission scoring one.vs comparison) and surveys (baseline vs endline), the first and second-order Rao-Scott corrections to the Pearson chi-squared test for proportions and two sample t-test for continuous variables were used.To establish significant differences between the groups  with the individual (subjects with febrile illness) as the first level and the cluster (village) as the second level. Akaike’s information criterion (AIC) value was used in the selection of the best-fit model.The mortality rates were computed as the ratio of the number of deaths and the mid-survey period population.The Ethical Review Committee, Department of Medical Research (Lower Myanmar) - No. 4/Ethics/2007 and the Ethics Committee, Prince of Songkla University, Thailand - EC 52-054-18-6-2 approved this study.Australian New Zealand Clinical Trials Registry ACTRN12610000706077.Characteristics of the volunteers and their assessment of the training workshop , availability of electricity (1/21 vs 0/17), availability of schools (20/21 vs 16/17), primary level schools (18/21 vs 13/17) and water wells as the main source of drinking water (19/21 vs 16/17).The trial intervention and comparison groups were similar in the main occupation being forest-related work . There was no significant difference in the duration of the intervention in different groups.All logbooks were available and submitted to the programme monthly. A total of 906 patients was registered by volunteers. Twenty six records were misplaced. Nine volunteers had at least one-month period without any RDT activity. It is not known whether this inactive period was due to his/her absence, RDT shortage or no case of fever.Logbook data are summarized in Table Of 381 positive results, eight received inappropriate chloroquine treatment, and seven inappropriate chloroquine treatment was given by a female volunteer. Of 498 negative results, eight were over-treated with AL, and five were given by an unlicensed practitioner who used RDTs from the dropout volunteer when PSI/Myanmar discontinued the RDT supply on a paid basis.Volunteers filled in case record forms for 89.9% (815/906) patients registered in their logbooks albeit leaving blank answers for a number of questions. Case recording data showed that 33.9% (260/768) patients were in the forest at the first episode of fever, 51.8% (342/660) cases self-medicated prior to consulting with volunteers, 1.5% (10/673) presented with poor health conditions and 40 were referred.P value from Mann–Whitney U test = 0.00001).When comparing timeliness of diagnosis from volunteer patient record forms with midwives’ logbooks, the number of days between the first day of fever and the day of consultation was significantly longer in intervention villages  than in villages with midwives  (The number of cases tested declined over the six-month period in both volunteer and MW groups but more sharply in the volunteer group. The positive test rate was slightly higher in the MW group. These rates fluctuated over time Table .vs comparison). However subjects in the intervention group were less likely to be Bamar and Buddhist. At endline, the proportion of children was greater in all three groups when compared to the baseline. In all three groups, over half of the patients were solely engaged in forest-related work or also working as farmers.As seen in Table vs endline mortality could not be compared because of differences in data collection procedures. Death rates were not different in the two groups in both baseline and endline surveys. Fever-related deaths accounted for approximately one third of all deaths, while one fifth was due to probable malaria. The proportion of deaths from fever and probable malaria were lower in the midwife villages , 116 (13.4%) reported receiving blood test-based treatment, 89.1% were tested by RDT and 68/116 (58.6%) reported the detection of malaria parasites. Anti-malarial treatment was prescribed to users without a blood test  by volunteers in two cases in the intervention group, by local health staff in five cases in the MW group and by unlicensed practitioners in 28 cases for all three groups .Results of the malaria transmission knowledge assessment with the WHO-modified questionnaire were low throughout the study. All mean scores were below three out of the maximum possible score of eight.The percentage of villagers with simple mosquito nets was almost 100% and the use of impregnated bed nets was low (<35% at the endline) in all groups. Compared to baseline, the number of villagers who impregnated their bed nets had significantly increased at endline in the intervention group while it significantly decreased in the comparison group. Both ownership of an impregnated bed net and sleeping under a bed net were significantly greater in the intervention than the comparison group.Results of multivariable analysis suggest that villages with a midwife or a volunteer had a greater chance of receiving a blood test than those without either. The odds of receiving RDT in subjects served by the two types of provider were not significantly different. Middle-aged men and those with impregnated bed nets received a malaria blood test more frequently than other groups Table .The intervention resulted in higher RDT coverage in the treatment villages, although an increase was also observed in the comparison village through contamination (receiving a test from the volunteer). The number of patients tested by volunteers declined in contrast to the stable number of patients tested by midwives. In all villages, timeliness of the intervention did not comply with WHO guidelines (over three days after onset of fever). There was no significant difference between groups in overall and probable malaria mortality rates. The low knowledge of patients was not improved by the intervention. However, the increase in the use of bed nets and reduction in the prevalence of fever observed was significant in the intervention group only.This study was based on cluster randomized controlled trial design that could minimize potential bias and confounders. Blinding used in randomized controlled trials on drug treatment was not possible because the training must be done openly. Exclusion of villages with security problems and those with too small population was unavoidable and it can be a limitation of this study. However, given that this is a relatively small proportion of the population in the study, the limitation should be of serious concern. The results of the randomization process were not perfect as mean distance from home of the villagers to the health facility in the comparison group was around 1 km, far more than that of the intervention group. However, the results of multivariable analysis showed that the outcome was not significantly influenced by the distance and so the distance could not explain the difference between the intervention and the comparison groups.The higher coverage of RDT after intervention may be explained by the increased availability of RDT, improved knowledge of free treatment and a low contamination (receiving RDT from volunteers) in the comparison group. The lower coverage of villagers in the comparison and intervention groups than in the midwife group may result from a number of factors: midwives had served villagers for longer, non-receipt of training in two intervention villages, unavailability of RDT service from the dropouts and shortage of RDT supply in the intervention group. The findings on the uneven availability of the programme are consistent with findings from similar volunteer interventions in Laos and Zambia ,27.While low coverage was observed in this study, higher coverage (40%) found in a previous study may be explained by the fact that half the subjects were from Bago Township where a VBDC office is located, that RDT facilities were available in most villages and hospitals in a few . In otheBoth the coverage of testing and time to blood test were low in all three groups. Such delays can lead to severe malaria  with treThe malaria mortality rates in this study are higher than statistics reported for most Southeast Asian countries. The rates in the intervention and comparison villages were greater than the overall malaria mortality rate  for Myanmar, which is comparable to the midwife group . The lowPossible reasons for the absence of difference in overall and probable malaria mortality  may include the relatively small study population, short follow-up, low coverage, insufficiently high endemicity for high mortality or a lack of malaria immunity among villagers. Declines in mortality have been reported in Thailand , EthiopiA co-intervention can potentially present a problem for the intervention trial. Using a cluster randomized design, the proportion of villages receiving impregnated bed nets was comparable in both groups, suggesting therefore that the co-intervention was not a likely confounder. While the co-intervention was spread similarly in both study groups, the use of impregnated nets was higher after the intervention, as was the knowledge of facilities for blood test and free malaria treatment. The training programme may have improved the volunteers’ communication skills to participate in impregnation of bed nets. In contrast, the decrease in the ownership of impregnated bed nets in the comparison group may result from the villagers’ knowledge of the six-month lifespan for bed net impregnation to remain active against a longer study period. Despite the presence of volunteers, ownership of impregnated bed nets in the study villages was much lower than that reported in studies from Ethiopia , ThailanThe number of patients tested by volunteers showed a declining trend. The seasonality of malaria could be a possible cause as malaria normally peaks in June-July and the intervention programme was launched in May. The greater decline in the volunteer group than the MW group may result from a possible unavailability of RDT facility, the absence of volunteers or a shortage of RDT in the volunteer group; an actual reduction in the number of fever cases could not be excluded. Volunteers’ absence may be seasonal, work- or study-related or the result of decreasing motivation over time for being unpaid. Shortages of RDT were due to delays in delivering supplies to volunteers, resulting from frequent staff transfers at the township level, limited numbers of malaria staff at the township level and the lack of budget for supervisory visits. The shortage of drugs supplied to community health workers in Zambia was found to be associated with a loss of reputation and subsequently low performance . While mMore male patients with fever were reported as were higher RDT positive rates than in females. These differences may be the result of work in the forest leading to increased exposure to malaria or other factors, such as a vulnerability to particular strains of the malaria parasite . In thisCompared to volunteers, midwives tested fewer younger people even though test results show a high positive rate. This may be explained by the integrated role of midwives in overseeing maternal and child health related issues. Children and young people are known to be more vulnerable to severe consequences from malaria and need more attention ,52.Other studies in Nepal , Kenya , Peninsu® RDT has been recommended in Myanmar for its high sensitivity and high prevalence of P. falciparum in the country. The findings in this study show high rates of P. falciparum malaria (43% of cases tested by volunteers and 49% by midwives). The limitation of Paracheck-Pf® RDT in detecting P. vivax malaria does not answer the question of the magnitude of non-fatal malaria. Presumptive treatment for a negative result with chloroquine does not eradicate P. vivax malaria. A 14-day primaquine regimen was not prescribed to avoid possible, serious adverse reactions. This could lead to a predominance of P. vivax malaria in this area in the future. It is hoped that with technological advances RDT will also become available for P. vivax malaria allowing them to be treated simultaneously.Paracheck-PfThis study demonstrates that the coverage of RDT can be improved by implementing a volunteer programme. However, the use of RDT was still low and that tests were not performed in a timely manner and that the number of patients tested declined over time. The use of impregnated nets and the knowledge of malaria were found to be low in all groups. The supervision component of the programme needs to be strengthened for quality control and sustainability of the programme.(MW): Midwives; (RDT): Rapid diagnostic test; (ACT): Artemisinin-based combination therapy; (AL): Artemether-lumefantrine; (VBDC): Vector Borne Disease Control team; (WHO): World Health Organization; (PSI/Myanmar): Population Services International/Myanmar; (SD): Standard deviation; (SE): Standard error; (IQR): Inter-quartile range; (OR): Odds ratio; (95% CI): 95% confidence interval.The authors declare that they have no competing interests.O, TM, SS, TW made contribution to conception and design of the study. VC made substantial contribution to revise the design. O, TM, SS carried out data collection and data cleaning. O and VC were involved in data analysis, interpretation and manuscript preparation. All authors read and approved the final manuscript. O is the guarantor of the paper.Training module on malaria volunteers. The training module on malaria volunteers is a translated version of the one available in Myanmar language.Click here for fileBackground information about volunteers and their assessment on training workshop.Click here for file


Plasmodium falciparum, poses a threat on critical transfusions in pregnant women and children. This study’s objective was to compare the performance of different malaria screening methods in blood donors in southern Benin, a malaria-endemic country.Malaria is a leading cause of mortality in southern Benin. The main causative agent, Plasmodium pLDH detection was assessed by an ELISA-malaria antigen test. Using crude soluble P. falciparum antigens, an ELISA-malaria antibody test detected anti-Plasmodium antibodies.Blood from 2,515 voluntary blood donors in Benin was collected over a period of 10 months in ethylenediaminetetraacetic acid (EDTA) tubes, which were then classified according to extraction time: long rainy season, short dry season, short rainy season, and long dry season. Microscopic examination was used to count parasites. Parasite density (PD) was expressed as the number of parasites per μL of blood. Pan Plasmodium carriage was 295/2,515 . Males had a higher infection rate (12.4%) than did females (8.8%). Parasite density was very low: between seven and100 parasites per μL of blood was reported in 80% of donors with parasitaemia. Three Plasmodium species were diagnosed: P. falciparum in 280/295 patients (95.0%), Plasmodium malariae in 14/295 (5.0%), and Plasmodium ovale in 1/295 (0.34%). Malaria prevalence in donors was higher during the rainy seasons (13.7%) compared with the dry seasons (9.9%). The use of a highly sensitive assay enabled pan Plasmodium pLDH detection in 966/2,515 . Malaria antibody prevalence was 1,859/2,515 . Donors’ antigenaemia and antibody levels varied significantly (P <0.05) over the course of the four seasons. The highest antigenaemia rate 323/630 (51.3%), was observed during the short rainy season, while the highest antibody prevalence, 751/886 (84.7%), was recorded during the long dry season.Among the 2,515 blood donors  screened, the rate of asymptomatic Plasmodium can transmit malaria to donation recipients. Malaria diagnostic methods are currently available, but the feasibility criteria for mass screening in endemic areas become preponderant. Detection of the pLDH antigen seems to be an adequate screening tool in endemic areas, for this antigen indicates parasite presence. Routine screening of all donated blood would prevent infected blood donations and reduce P. falciparum transmission in critical patients, such as children and pregnant women. This tool would also decrease medical prophylaxis in donation recipients and contribute to lower Plasmodium resistance.Blood donations infected with  Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, and Plasmodium knowlesi) may be transmitted via blood transfusion [Plasmodium prevalence among blood donors may reach record levels of 51.50% [Transfusion-induced malaria was first reported in 1911 [nsfusion . Blood insfusion . Blood tnsfusion ,4. As mansfusion , this wonsfusion ,7. Transnsfusion , Brazil nsfusion , and thef 51.50% ,13 usingf 51.50% .Anopheles gambiae, is the most widespread and difficult to control [P. falciparum. In Benin, 37% of adult health facility visits and 41% of those by children under five years old are malaria-related. Malaria is the leading disease affecting communities, with pregnant women and children under five years old being the most vulnerable. The incidence of uncomplicated and severe malaria was 139 per 1,000 inhabitants in 2006 [Plasmodium transfusion is expected to increase over the next few years in unprotected patients, such as pregnant women or children. Blood transfusion is the third transmission path of Plasmodium, and there is still no consensus on TTM preventative measures to be taken in tropical settings [Plasmodium[In Benin, as in other tropical developing countries, the high demand for blood donations due to increased road accidents, pregnancy-related haemorrhages and child anaemia enhances the risk of TTM. Benin’s humid tropical climate, which has two rainy and two dry seasons, favours malaria transmission over the course of eight months, with 58 infectious mosquito bites per man per year . The mos control . The maj in 2006 . Self-me in 2006 . The sigsettings . Malarialasmodium,20. Nevelasmodium-23. The lasmodium. TherefoBenin is characterized by tropical rainforest vegetation, Sudanese climate type with an annual rainfall of about 1,600 mm, and an atmospheric temperature of 32°C. There are four distinct seasons: two rainy and two dry. This descriptive, transversal study was conducted in Benin over ten consecutive months in six departments, which grouped into three pairs of Departmental Blood Transfusion Centres (DBTC): Atlantique-Littoral,Oueme-Plateau, and Mono-Couffo. They serve as referral centres for south Benin blood transfusion services. This study was approved by the Direction of Benin National Blood Transfusion Agency and the Research Ethics Committee of the Republic of Benin. Approval was granted under the following conditions: donor anonymity must be maintained, good laboratory practice quality control must be ensured, and every finding must be treated with utmost confidentiality and used only for this research purpose. Biological analyses of the samples were performed in the biochemistry and molecular biology laboratory in Cotonou (Benin) and the Institut de Parasitologie et de Pathologie Tropicale de Strasbourg (IPPTS) (France).Individuals considered healthy who visited the three DBTCs for blood donation between May 2009 and March 2010 were considered eligible for the study. A total of 2,515 donors and regular voluntary blood donors were enrolled over a period of ten months, which was divided into a long rainy season (LRS) from May to July, a short dry season (SDS) from August to September, a short rainy season (SRS) from October to November, and a long dry season (LDS) from December to March. Donors were selected according to medical screening criteria: no fever, no clinical evidence of progressive disease, weight above 50 kg, age between 18 and 60 years old, or 65 years old for regular donors. Last blood donation had to have occurred within the previous four months for women and three months for men. A more systematic questionnaire was introduced providing the full name, age, sex, address, and telephone of each individual. Malaria-related questions were also included: date and confirmation via clinical manifestation of malaria episodes that occurred during the year, subsequent treatment and prevention measures taken against malaria. Each study participant signed an informed consent. Overall, 7 mL of blood were collected in EDTA tubes, with 1 mL transferred into a microtube and the remaining 6 mL centrifuged at 4°C, plasma being collected into a new microtube. The tubes were stored at −20°C and transported to the IPPTS laboratory in solid carbon dioxide for antibody and pLDH antigen detection.A thick and a thin smear were performed on slides for each Benin blood donor. The thick film was dehaemoglobinized using distilled water at pH 7.2, and then thick and thin blood films were stained using a May-Grünwald-Giemsa (MGG) stain. The microscopic reading and parasite density calculation were performed by two laboratory technicians, and in the case of uncertainty, also by a third one. Parasite count was made on the basis of 500 parasites per 1,000 leukocytes. The reading was stopped after 500 parasites were counted, even though the 1,000 figures of leukocytes were not reached. Parasite density (PD) was expressed as the number of parasites per μL of blood.Plasmodium species, but it possesses species-specific isomers [Plasmodium in blood, and is used for screening in malaria-endemic countries. The pLDH antigen detection was performed by a sandwich enzyme-linked immunosorbent assay (ELISA), notably an ELISA-malaria antigen test  that detects pLDH via immunocapture. The apDia Antigen ELISA is an in vitro diagnostic immunoassay (IVD) for the qualitative determination of Plasmodium spp. LDH in blood samples. The apDia Malaria antigen test can be used in order to detect the malaria pLDH antigen of any of the four species in blood samples. The test was performed according to manufacturer recommendations: pour 100 μL of ready-to-use lysing buffer into each well; add 50 μL of reconstituted positive control to one well and 50 μL of negative control to triplicate well; add 50 μL of homogenized fresh whole blood sample into corresponding well; incubate for 60 min at 37°C under continuous gentle shaking conditions; drain the wells via aspiration, and fill them completely with 350 μL of washing solution; allow the wells to soak for 1 min before washing again five times; pour 100 μL of conjugate 1 solution into each well, and incubate the plate for 30 min at 37°C; wash the wells five times and pour 100 μL of conjugate 2 into each wells. Incubate the plate for 15 min then wash the wells five times and pour 100 μL of chromogenic solution into each well; incubate the plate for 15 min at 37°C; add 50 μL of stopping solution to all wells and read absorbance of each well at 450 nm with reference wavelength of 620 nm within 15 min. Test results were interpreted as follows: the optical densities of positive control (ODpos) must be >0.500 and the average OD of negative control (ODneg) <0.100. The ODneg was used to calculate the cut-off by multiplying its average value by three. The antigen index (Ag Index) of each sample was calculated by dividing the OD value of the sample by the cut-off value. A sample was considered positive if the Ag Index was ≥1.0, indicating that the sample contained viable parasites. A sample was considered negative if the Ag Index was ≤0.8, indicating that there were no viable parasites in the blood, or that there was no Plasmodium multiplication because of anti-malarial drug intake. A sample was considered inconclusive if the Ag Index was between 0.8 and 1.pLDH is a glycolytic pathway enzyme secreted by the different  isomers . The pLD isomers . TherefoP. falciparum, 19 for P. ovale, six for P. malariae, and two exhibited a mixed infection . This panel was used to assess the sensitivity of the ELISA antigen detection test compared to microscopy.The whole blood biobank collected from 266 malaria patients who had returned from endemic countries, positively diagnosed and referred by Strasbourg University hospital was tested. Among them, 239 were positive for Blood donor samples were collected at the French Etablissement Français du Sang d’Alsace (EFS Alsace). Based on a medical questionnaire, donors were classified as “malaria-risk blood donors”  if they had travelled to an endemic area in the previous four months, or “not-exposed-to-malaria blood donors”  if they had not travelled to an endemic area in the previous three years.P. falciparum was maintained in vitro using an adapted candle jar method for continuous culture [The 3D7 strain of  culture . ParasitCulture with 65% ring-stage parasites and 35% schizonts was used. Infected RBCs from the continuous culture at 1% parasitaemia determined on thin blood film by microscopy were serially diluted in uninfected RBCs by incubation with low agitation at 37°C. Dilution ranged from 20,000 infected RBCs (iRBCs)/μl to 0.1 iRBCs/μl. For microscopy examination, thin blood films were made from each dilution after careful suspension, while thick blood films were made for the lower parasitaemia. Three slides were prepared for each dilution and read by two microscopists in a blind manner. Parasitaemia was determined by counting fields with 100 RBCs, and the total level was expressed in μL of blood corresponding of 5.106 RBCs/μL. A linear regression curve based on mean parasitaemia levels was established in order to determine the parasitaemia, and a standard curve was generated using recombinant pLDH to assess the correlation between parasitaemia and pLDH concentration.P. falciparum pLDH protein was inserted in the expression vector pMAL C2X  containing the maltose binding protein (MBP) fusion partner sequence. The protein was expressed in Escherichia coli and purified by affinity chromatography on amylose resin. The protein was used to generate the standard curve.A synthetic gene  encoding for Plasmodium antibodies present in serum or plasma samples to antigens immobilized on 96-well plates, with the antigen being an extract of in vitro P. falciparum culture (3D7 strain). Due to antigenic community, antibodies to other Plasmodium species may be detected. The diluent solution containing PBS and Tween 0.1%, (125 μL) was poured into each well, followed by 25 μL of test plasma. On the same plate, 25 μL positive control and negative control were poured in single and triplicate wells, respectively. The plate incubated for 60 min at 37°C before being washed five times. One hundred μL of horseradish peroxidase-conjugated rabbit anti-human IgG polyclonal antibodies  were added to each well, and the plate incubated for a further 30 min at 37°C. The wells were washed five times, and 100 μL of TMB plus substrate solution (tetramethylbenzidine)  were added to each well. The plate was covered and incubated in the dark for 15 min at 37°C. Finally, 50 μL of 0.5 M sulphuric acid was added to each well, and absorbance was read within 15 min at 450 nm, with a reference wave-length of 620 nm. Test validation required that the positive OD be >0.500 and the negative OD≥0.200. The cut-off value was calculated by multiplying the negative control wells’ average OD by four. The antibody (Ab) index of each sample was calculated by dividing its OD value by the cut-off value. The sample was considered positive if the Ab index was >1.0, equivocal if the Ab index was between 0.8 and 1.0, and negative if the Ab index was ≤0.8. The test was not able to distinguish between antibodies directed to either P. falciparum, P. vivax, P. malariae, or P. ovale.Antibody screening was performed using a homemade ELISA-antibody test. The serological ELISA test was derived from a commercial assay exhibiting high specificity and analytical sensitivity, namely 96.7 and 93.1%, respectively . The tesThe data was analysed by the chi-squared, Bartlett's chi-squared, and Kruskal-Wallis tests using Sigma Plot 2001 software  and Epi Info version 3.5.1. The results were expressed as mean ± standard deviation. Statistical significance was reached if P <0.05, with a 95% confidence interval (CI).P. falciparum were missed, five of them were treated for malaria before the blood examination and the four remaining patients did not provide any information’s regarding a previous treatment. Seven cases of P. ovale were also missed may be due to the fact that the monoclonal antibodies for pLDH fail to detect some P. ovale strains. However this point was not explored in our study and not discussed. The antibody detection test was able to detect only 220 cases , with a linear regression line and 0.977 correlation coefficient. The detection limit was 1 parasite/μL for the pLDH ELISA, which corresponds to 0.08 ng/ml of recombinant pLDH. The detection threshold with the standard curve using the recombinant pLDH was 0.125 ng/mL, the curve being linear up to 2.5 ng/mL Figure .versus 488/2,515  women. Among the donors, 1,989/2,515 slept under a mosquito net . The majority of donors, namely 1,521/2,515 , reported having had episodes of malaria, and the predominant clinical prior malaria expression was a single suspected access in 1,387/2,515 , whereas 51 donors  reported a severe attack requiring hospitalization in the last year. In total, 83 donors  had both single and severe malaria attacks, whereas 994 donors (39.52%) reported having no malaria attack during the preceding year. Among the 1,521 donors with suspected malaria episodes, anti-malarial treatment was administered to 509 (33.46%) in health facilities during the last malaria attack, whereas 1,012/1,521 (66.53%) were treated as outpatients. Most blood donors did not receive chemoprophylaxis but performed a sort of prevention when they experienced “signs of malaria”. Self-prevention against malaria was undertaken by 1,537/2,515 donors (61.11%) who took anti-malarial drugs or herbal tea, whereas 978/2,515 (38.88%) did not undertake any prevention. The drugs used were quinine 567/2,515 , artemisinin-based combination 510/2,515 , herbal teas 336/2,515 , and Fansidar® 124/2,515 .Blood samples from 2,515 donors were collected over a ten-month period covering four seasons: LRS, SDS, SRS, and LDS. The samples were distributed as follows: 590/2,515 (23.5%) in LRS, 409/2,515 (16.3%) in SDS, 630/2,515 (25.0%) in SRS and 886/2,515 (35.2%) . Three Plasmodium species were detected: P. falciparum was identified in all infection cases, P. malariae in 14 mixed infection cases (5.1%), and P. ovale in one mixed infection case (0.34%)  exhibited a parasite density between seven and 100 parasites per microliter (p/μL), with 0.3% exhibiting a density >5,000 p/μL  (95% CI: 72.1-75.6%). Equivocal results were found in 13.1% of samples , and there were no detectable antibodies in 327 donors   (95% CI: 36.5-40.3%), with equivocal results  considered as positive for the calculation. The prevalence of pLDH antigen confirmed by microscopy was 230/966 (23.8%). The only sample that was positive by microscopy but negative by pLDH detection was a 6/2,515  Table . InteresPlasmodium LDH ELISA was shown to exhibit optimal performances as to both PPV and NPV. The assay was able to detect one parasite per μL, which is the lowest detectability reported in the scientific literature to date. This detection threshold is much lower than that of the already commercialized rapid test [PfHRP2 [in vitro lower amounts of P. falciparum and other species, would thus be an interesting tool to detect the presence of Plasmodium in a given blood donor population. However the reagent failed to detect seven cases of P. ovale out of 19 cases. This defect could due to the incapability of the assay to detect the pLDH from some strains of P. ovale[Plasmodium LDH ELISA was evaluated in a large blood donor population from southern Benin. The test results were compared to those obtained using microscopy and to those pertaining to malaria-antibody prevalence. The study aimed to determine whether pLDH detection was sensitive enough to identify low parasitaemia levels, which are frequently the case in asymptomatic malaria blood donors, and to assess the prevalence of positive pLDH blood donations in southern Benin.In this study, a commercialized pan pid test , which i [PfHRP2 . The aut P. ovale. The useP. falciparum (97.10%) and P. malariae (2.00%) [P. falciparum and 4.7% prevalence of P. malariae. The overall prevalence of asymptomatic Plasmodium carriage here was 11.72%. Plasmodium falciparum parasite density proved to be very low and highly variable. Two-third of these positive donors had a parasite density between seven and 100 parasites per μL. Reported prevalence rates in Benin appear to be similar to those reported by some authors in neighbouring Nigeria, 10.2% [P. falciparum was identified in all infected cases, 5.0% of which were mixed P. malariae infections. These figures are in line with the reported presence of P. falciparum in all infected cases and the 2.3% rate of mixed infection in Nigeria [This study enrolled 2,515 blood donors from six southern Benin departments, with similar malaria epidemiological features. These departments exhibit a wet tropical climate, with two rainy seasons and two dry seasons. In these regions, malaria transmission is permanent, with an increase during the rainy season . The mos (2.00%) . The figa, 10.2% , or in Ga, 10.2% . In this Nigeria , 55% in  Nigeria ,23, and vs 14.6%). In a study conducted in Colombia [Plasmodium ssp. than females, 59 and 41%, respectively. Nevertheless, in a 1994 publication by Vlassoff et al.[The study findings highlighted a higher infection rate in male blood donors compared to female ones  in the study’s blood donor populations. This prevalence was similar to that reported for Senegal, with 65.33% being positive and 6.86% equivocal . Therefoet al. in Senegal, who reported a detection rate of 0.53% for pLDH, with a 2.23% rate of doubtful cases [In this study, 38.4% of the blood donors were tested positive for pLDH antigen, with a 4.2% rate of equivocal results. The pLDH antigen detection results differed from those found by Diop ul cases . HoweverPlasmodium screening in blood donation, which was not performed in Benin due to costs and lack of technique reliability, still keeping in mind that blood donors who report an undiagnosed and untreated febrile illness consistent with malaria should be deferred until asymptomatic and off the treatment. In a recent cost effectiveness analysis study, Rajab et al.[The questionnaire’s effectiveness helped to identify certain risk factors associated with pLDH antigen carriage, such as blood donations during high transmission periods. Based on the study results it was noticed that seasons influenced both microscopic and pLDH prevalence. Others factors likely impact these markers, such as the malaria prevention method used by blood donors, i.e. drugs, herbal teas, etc. The limits of selecting blood donors through medical questionnaires were highlighted by the study findings, as 38.4% positive donors for ELISA pLDH had met the selection criteria and gave their blood. It is, therefore, logical to propose implementation of Plasmodium could be an interesting tool for blood donation qualification in order to ensure blood safety in malaria-endemic areas. One of the test’s advantages is that false-positives are exceptional, unlike the HRP2 (histidine-rich-protein)-based tests, which can remain positive for two weeks after parasite clearance. HRP2 may also be absent in malaria infection cases. pLDH antigen ELISA detection in the blood is efficient when there is a parasite [P. ovale infection cases the assay failed to detect them. Similarly, if the donors had taken self-treatment measures prior to blood donation, malaria infection was masked and pLDH detection failed. In conclusion, routine screening of all donated blood would prevent infected blood donation and increase the donation’s safety for fragile recipients, such as children and pregnant women. It would also be instrumental in reducing unnecessary medical treatments in recipients and contribute to lower Plasmodium resistance. The main problem remains the feasibility of rejecting positive donations in term of blood availability in endemic area where blood transfusion needs are presently increasing.Malaria and transfusion is a “neglected subject coming back to the fore front”, as recently discussed by Allain . The micparasite . Some piThe authors declare that they have no competing interests.PSA, CDL, AS and EC designed the study, and performed the acquisition, the analysis and the interpretation of data. NWC, SP, TA, CDA, LA and AB were involved in the acquisition, the analysis and interpretation of data. All authors have given a final approval to the manuscript.


Plasmodium falciparum malaria. However, there is little information on the anti-malaria immune profile of the population in the endemic regions of the country.In Ethiopia, the general population is vulnerable to unpredictable epidemics of P. falciparum blood-stage vaccine candidate GMZ2 and its subunits (Glutamate-rich protein (GLURP-R0), merozoite surface protein 3 (MSP3); as well as IgG subclasses against GLURP-R0 and MSP3.The study was designed to investigate the nature of humoral immune response to malaria in two ethnic groups in two endemic localities: Shewa Robit in north, and Boditi in south Ethiopia which are characterized by varying levels of malaria transmission and altitude. In a cross-sectional study, the study participants were diagnosed for malaria infection microscopically and by the rapid diagnostic test (RDT). Sera were tested by using enzyme-linked immunosorbent assay (ELISA) for total immunoglobulin (Ig) G against P. falciparum were detected in Boditi, all Shewa Robit study participants had no detectable P. falciparum infection. In both localities, total IgG prevalence and levels to GMZ2 were significantly higher than the response to the component domains indicating the strong recognition of GMZ2 by antibodies acquired through natural exposure. Total IgG and subclass prevalence and levels were higher in Shewa Robit than Boditi, suggesting difference in the intensity of malaria transmission in the two localities and/or genetic differences between the two populations in their response to the antigens. In both study sites, IgG subclass levels to GLURP-R0 were significantly higher than that to MSP3 for all corresponding subclasses in most individuals, indicating the higher relative antigenicity and probably protective potential of GLURP-R0 compared to MSP3. Against both GLURP-R0 and MSP3, the ratio of cytophilic to noncytophilic antibodies was >1 in the majority of the study participants, in both study sites, suggesting the induction of protective (cytophilic) antibodies against the two antigens. Analysis of age-related pattern in antibody levels against the antigens showed a positive association with increasing age.Whereas 23(8.6%) blood smear-positive cases for P. falciparum GLURP-R0 and MSP3 separately as well as in a fused form in GMZ2 are readily recognized by the sera of the study populations. The significantly higher antibody prevalence and level detected against GMZ2 compared to either of its subunits separately, in naturally exposed populations, suggests the synergistic effect of GLURP-R0 and MSP3 and that GMZ2 could be a more relevant blood-stage malaria vaccine candidate than the individual components. Detection of high-level antibody responses in non-febrile, smear-negative individuals may possibly be an indication of a low-grade, asymptomatic sub-microscopic infection in the induction and maintenance of high-level malaria immunity. Plasmodium falciparum is the most widely distributed and deadly species. Drug resistance of the parasite and insecticide resistance of the vector prove the fight against the disease is challenging. An effective malaria vaccine would be integrated into existing control strategies and make malaria elimination/eradication plans more feasible. Various malaria candidate vaccines are at different clinical trial levels; until now only one of them, the RTS,S vaccine candidate, completed Phase III [Globally, over 225 million malaria cases and 781,000 deaths occur annually [hase III ,4. Indivhase III .P. falciparum[P. falciparum isolates, is the C-terminal region, representing amino acids 212–318 [Lactococcus lactis from genetically coupled P. falciparum GLURP-R0 and MSP3212-380[While MSP3 is an erythrocytic-stage protein glutamate-rich protein (GLURP) is expressed in both pre-erythrocytic and erythrocytic stages of alciparum. A 220-kalciparum. A major 212–318 . GMZ2 isP3212-380. Phase IP3212-380 and natuP. falciparum and P. vivax are the dominant malaria parasites in Ethiopia accounting for about 60% and 40% of total malaria cases, respectively. An. arabiensis is main disease vector, while An. pharoensis, An. funestus and An. nili occasionally transmit the disease in certain areas of the country [The study was carried out in Shewa Robit, a town in north-central Ethiopia located about 225 km north of Addis Abeba with geographic coordinates 10°0′36″ N latitude and 39°54′10.8″ E longitude and Boditi located in south western Ethiopia, about 370 km away from the capital having coordinates of 7°2′16.8″ N latitude and 37°53′16.8″ E longitude .The study was approved by the Ethics Review Committee of Department of Biology, Addis Abeba University and had a national ethical clearance from the Ethiopian Federal Ministry of Science and Technology. Informed consent was obtained from adult participants and parents/legal guardians for children prior to sampling. Malaria smear-positives were treated free of charge as per the national treatment guideline  and 2009 (Boditi), both in the dry season when there was minimum malaria transmission. Five ml of venous blood was collected from each participant. Malaria RDT using CareStart™ malaria HRP2/pLDH(Pf/pan) Combo Cassette Test  was carried out as per the manufacturer’s instruction. Thick and thin blood smears were prepared and microscopically screened for Total IgG was measured against GMZ2, GLURP-R0 and MSP3, and IgG subclass levels for GLURP-R0 and MSP3. All ELISA experiments were performed following a previously published test protocol . In briep<0.05, 95% confidence intervals (CI) were considered significant.The data was double entered into separate Microsoft Excel spreadsheets and checked for overall correctness and normality. As mean antibody levels were not normally distributed, nonparametric tests were used for analyses. Fisher’s exact test was used to test the significance between differences in antibody positivity rates for the antigens within and between the two study sites as well as between age groups, slide-positives and –negatives and between IgG subclasses. To test the difference in OD levels between more than two groups the Kruskal-Wallis test was used. Differences between median OD values for each antigen between two groups (pairwise) were analyzed by Mann–Whitney U (Wilcoxon rank-sum) test. Multiple comparisons were used to assess between which particular age groups (for more than two age groups) the significant differences existed. Spearman rank correlation was used to test the correlation between antibody level to each antigen and the age-related pattern of antibody level. Age-stratified analysis was followed for comparisons between the two study sites. Analysis was done using SPSS version 17.0 for Windows . Two-sided Plasmodium infection at the time of sampling. A total of 265 serum samples was collected from residents of Boditi with mean age of 23 years. The highest age was 80 and the lowest was one year. Table P. falciparum. Twelve asymptomatic infections were detected making the total number of positives 23(8.6%). Only one P. vivax infection was detected by microscope.Some 228 serum samples were collected from participants in Shewa Robit. The mean age was 28 years and there were no individuals below five years of age (range 5–80 years). All participants were asymptomatic and had no microscopically or RDT detectable p < 0.001). Anti-GLURP-R0 total IgG positivity rate was higher than those to MSP3 in Boditi and the reverse was observed in Shewa Robit but the differences were not significant in both cases. Total IgG positivity rates were higher in Shewa Robit than Boditi for all the three antigens though all the differences were not significant. Total IgG levels to GMZ2 were significantly higher than that to MSP3 or GLURP-R0 in both sites (p <0.001). Total IgG levels to GLURP-R0 were higher than that to MSP3 but significant difference was observed only for Shewa Robit (p = 0.012). For all the three antigens tested, total IgG positivity rates were higher in Shewa Robit than Boditi though the differences were not significant. Total IgG levels were significantly higher for GMZ2 (p = 0.013) and GLURP-R0 (p = 0.021) in Shewa Robit than Boditi between age-stratified groups.Antibody levels to GMZ2, GLURP-R0 and MSP3 were at detectable level in most individuals in both sites  of serum samples were IgG positive against GMZ2, GLURP-R0 and MSP3 only 6.8% did not have a detectable IgG level against any of the antigens.p < 0.01) , MSP3 (p = 0.015) and GLURP-R0 (p <0.001). Levels of total IgG also showed an age-dependent increase with significant differences between all the three age groups for GMZ2, between under-four years and above 14, and between 5–14 years and above 14 for MSP3 and GLURP-R0 (p <0.001)  and IgG2 (p < 0.001) antibody levels to MSP3 were significantly higher in Shewa Robit than Boditi between age-stratified groups. More samples were positive to GLURP-R0 but not MSP3 for all subclass antibodies in both sites. Also, antibody levels were significantly higher for GLURP-R0 than MSP3 for IgG1, IgG2 and IgG3 (p < 0.001) in both study populations. Although it may not necessarily reflect the real antibody concentration level, in terms of OD values in the majority of the study populations cytophilic subclasses were dominant over the noncytophilic ones for both antigens in both study sites.Positivity rates of IgG1 and IgG3 to GLURP-R0 and all subclasses to MSP3 were significantly higher in Shewa Robit than Boditi between age-stratified groups Table . SimilarThe significantly higher total IgG prevalence and level detected in both study sites of the present study for GMZ2 than either of its two component domains indicates the enhanced antigenicity of GMZ2. The findings suggest that people in endemic areas have high antibody levels to the GLURP and MSP3 components of GMZ2 antigen, which is part of the GMZ2 vaccine candidate, in the absence of microscopically detectable infection. The higher total IgG and subclass levels against GLURP-R0 than MSP3 in both study sites indicates the better antigenicity of GLURP-R0 compared to MSP3. The findings are in line with data from pre-clinical and clinical evaluations of the GMZ2 vaccine candidate. Pre-clinical studies in mice  and monkInitial clinical trials in malaria-naïve  and natuThe inter-individual variability in antibody response to a specific antigen observed among the study participants was also reported from other malaria endemic areas . In the P. falciparum vaccine antigens and how they are maintained remains poorly defined. In this study, in the context of heavily reduced transmission, strong immune responses were recorded for all of the antigens in most study participants. This suggests the stability of malaria immunity. The data provided additional evidence in support of earlier observations that antibodies can last for years in the absence of significant boost infections [Currently, knowledge on the durability of antibody responses to fections ,19.After one year, antibody level to GMZ2 remained high in the first clinical trial in malaria-naïve adults although it was lower compared to the level on day 84 . In the The present study suggests that both antigens, GLURP-R0 and MSP3, are important for the induction of protective antibodies against malaria from natural infections providing additional rational for combining the two antigens in a hybrid vaccine formulation. Thus GMZ2 has high potential as a candidate vaccine compared to either GLURP-R0 or MSP3 candidate vaccine antigens. However, it remains unclear whether the GMZ2 vaccines would be effective in the heterogeneous epidemiological settings of Ethiopia as there is evidence that vaccine-induced antibodies may not be as potent as those naturally acquired . But theThe overall dominance of cytophilic IgG subclasses over the noncytophilic shows the functionality of GLURP-R0-specific IgG subclasses explaining the ‘better protected’ status of Shewa Robit participants who were asymptomatic and microscopy negative. The dominance of MSP3-specific IgG1 and IgG3 isotypes in Boditi sera goes in agreement with studies from other settings ,24. In bin vivo. Cytokines and B cell activators are involved in inducing Ig class switching in a model system [Factors that control the preferential induction of polarized subclass responses are not well understood l system . It was l system  and allel system . Furtherl system  especiall system . Malarial system .P. falciparum infection in Boditi sera. This suggests that smear-negative individuals were relatively protected due to their high antibody levels. The prevalence of IgG to MSP2 serogroup antigens in a seasonal transmission area correlated positively with presence of parasitaemia at the time of sampling [P. falciparum in blood and the level of parasitaemia [The age-related increase of antibody levels observed for the antigens in Shewa Robit is in agreement with other studies ,31,32. Asampling . Such resitaemia .P. falciparum-specific responses were obtained for people living at elevations of 1,828 masl and less compared to those at 1,920 or above [The significantly lower falciparum-specific antibody level noticed in Boditi than Shewa Robit may be due to genetic differences between the two population groups, parasite strain variation in the localities, or level of malaria transmission. Boditi is a highland fringe at an altitude of 2,059 m above sea level (masl). The seasonality of malaria in highlands is more pronounced with characteristic lower immunity compared to warm hotbeds. A classical serological survey conducted in Ethiopian highlands indicated that high or above . TreatmeP. falciparum blood-stage antigens in the absence of microscopically detectable infection. Higher antibody prevalence and level detected against GMZ2, compared to its subunits individually, suggests that this antigen could be a more relevant blood-stage malaria vaccine candidate. Though a few sera did not show reactivity to GLURP-R0 and MSP3 in both study sites, most were positive for all IgG1, IgG2 and IgG3 subclasses suggesting that the antigens are readily recognized in the populations. The ratio of cytophilic to noncytophilic antibodies against GMZ2 subunits was >1 in the majority of the study population in both study sites implying the stimulation of functional IgG subclasses. The highest antibody prevalence and level were among adults and varied between antigens.Detection of high antibody prevalence and level in smear-negative individuals suggests that people in seasonal malaria transmission areas of Ethiopia acquire and maintain immune responses to ELISA: Enzyme-linked immunosorbent assay; AMA: apical membrane antigen; FMP: Falciparum malaria protein; GLURP: Glutamate-rich protein; Ig: Immunoglobulin; IRS: Indoor residual spray; IQR: Interquartile range; ITN: Insecticide-treated bed net; MSP: Merozoite surface protein; TMB: Tetramethylbenzidine; PBS: Phosphate buffered saline; HRP: Horseradish peroxidase; OD: Optical density; CI: Confidence interval; SD: Standard deviation; UK: United Kingdom; USA: United States of America.The authors declare that they have no competing interests.HM contributed towards the conception and design of the study; acquisition, statistical analysis and interpretation of the data; and drafting and revision of the manuscript. ME supervised laboratory work, interpreted the data and revised the manuscript. AA participated in laboratory investigations, interpreted the data and revised the manuscript. MT interpreted the data and revised the manuscript. BM supervised laboratory work, interpreted the data and revised the manuscript; BP conceived the study, participated in its design, supervised fieldwork, interpreted the data and revised the manuscript. All authors read and approved the submission of the manuscript for publication.


Plasmodium falciparum provide information on continued malaria transmission and potentially susceptible populations. Traditional techniques for measuring serological responses to P. falciparum antigens use plasma or dried blood spots (DBS). These invasive procedures pose a biohazard and may be unacceptable to communities if performed frequently. The use of oral fluid (OF) samples to detect antibodies to P. falciparum antigens may be a more acceptable strategy to monitor changes in population immunity.In formerly endemic areas where malaria transmission has declined, levels of population immunity to P. falciparum antigens. Optical density (OD) values from paired DBS and OF samples collected as part of a community-based survey of malaria parasitaemia were compared.An enzyme immunoassay was optimized to detect antibodies to whole, asexual stage Oral fluid and dried blood spot samples were collected from 53 participants in Southern Province, Zambia. Their ages ranged from 1 to 80 years and 45% were female. A statistically significant correlation  was observed between OD values from OF and DBS samples. The OF assay identified all DBS-confirmed positive and negative samples, resulting in 100% sensitivity and specificity.P. falciparum antigens. As OF collection is often more acceptable to communities, poses less of a biohazard than blood samples and can be performed by community volunteers, serological surveys using OF samples provide a strategy for monitoring population immunity in regions of declining malaria transmission.Oral fluid is a valid alternative specimen for monitoring changes in antibodies to  Plasmodium falciparum provide information on foci of malaria transmission and potentially susceptible populations [P. falciparum antigens pose a biohazard and may be unacceptable to communities if performed frequently [In formerly malaria endemic areas where transmission has declined, changing levels of population immunity to ulations . Serologulations ,2. Tradiequently .Oral fluid (OF) refers to crevicular fluid, the transudate from the crevice between the gum margin and teeth, collected from the mouth using an absorptive device . CrevicuP. falciparum antigens may be a more acceptable strategy to monitor changes in population immunity, particularly in regions of declining malaria transmission. However, no published evidence exists on the use of OF to detect antibodies to P. falciparum. The use of OF samples to measure antibody levels to P. falciparum antigens was validated in a region of declining malaria transmission in southern Zambia.The use of OF specimens to monitor changes in antibody levels to Anopheles arabiensis is the primary vector responsible for malaria transmission [The study was conducted in the catchment area of Macha Hospital in Southern Province, Zambia. Macha Hospital is approximately 70 km from the nearest town of Choma and the catchment area is populated by traditional villagers living in small, scattered homesteads. smission , which psmission .Satellite images were used to construct a sampling frame for the random selection of households. Permission from the chief and head of household were obtained prior to the study visits. Field workers obtained individual informed consent and a questionnaire was administered to each study participant to collect demographic information as well as information on prior malaria infections and treatment history. Blood samples were collected by finger prick and stored as DBS on filter paper . The cards were dried overnight and stored individually with desiccant in sealed plastic bags. OF samples were obtained from the same participants using Aware Messenger oral-specimen collection devices . Participants' gums were swabbed for one minute according to the manufacturer's instructions, and samples were transferred to a collection tube with the manufacturer's transport buffer. Samples were stored at room temperature. The study was approved the Johns Hopkins Bloomberg School of Public Health Institutional Review Board and the University of Zambia Research Ethics Committee.P. falciparum antigens with known positive and negative control samples  overnight at 4°C. The plate was blocked using 5% skim milk with phosphate buffered saline for one hour at 37°C. Serum and OF samples were plated in triplicate and incubated for one hour at 37°C, and peroxidase-labeled goat anti-human IgG was added to the plate for one hour at 37°C. ABTS was added to the plate for 15 minutes at room temperature before the optical density (OD) was read at 405 nm.An enzyme immunoassay (EIA) was used to detect antibodies to whole, asexual stage es Table . Sera weTo classify samples as seropositive or seronegative, test results were compared to a threshold OD level established as three standard deviations above the OD observed from filter paper spotted with serum from individuals who reported never having been exposed to malaria. OD values from paired DBS and OF samples were compared using Stata version 11.0 and the correlation coefficient was computed.2 = 0.63, P < 0.01) was observed between OD values obtained from OF and DBS samples . However, OD values obtained from OF samples were consistently lower than those obtained from DBS. The OF assay identified all DBS-confirmed positive and negative samples, resulting in 100% sensitivity and specificity ; DBS: dried blood spots; EIA: enzyme immunoassay; OD: optical density; OF: oral fluid; PBST: phosphate buffered saline with 0.05% Tween 20The authors declare that they have no competing interests.APC performed the enzyme immunoassays and drafted the manuscript. SC performed and supervised the enzyme immunoassays. TK designed the experiments, supervised the enzyme immunoassays and participated in the preparation of the manuscript. HH participated in the design and coordination of the data and sample collection. SM participated in the design and coordination of the study. PET participated in the design and coordination of the study. WJM conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.


To compare the performance of the Paracheck™ rapid diagnostic test (RDT) with microscopy for diagnosing malaria in hospitalised children.Children aged between 2 months and 13 years with fever were enrolled in the study over 1 year. A standard clinical history and examination were recorded and blood drawn for culture, complete blood count, Paracheck™ RDT and double-read blood slide.Salmonella bacteraemia (OR 3.89. 95% CI 2.27–6.63). In spite of high sensitivity, 56/2195 (2.6%) of true infections were Paracheck negative and 8/56 (14.3%) were in patients with >50 000 parasites/μl.Of 3639 children enrolled, 2195 (60.3%) were slide positive. The sensitivity and specificity of Paracheck were 97.5% (95% CI 96.9–98.0) and 65.3% (95% CI 63.8–66.9), respectively. There was an inverse relationship between age-specific prevalence of parasitaemia and Paracheck specificity. In logistic regression model, false-positive Paracheck results were significantly associated with pre-admission use of antimalarial drug , absence of current fever  and non-typhi Salmonella infection. While Paracheck was highly sensitive, some high-density infections were missed. For children with severe febrile illness, at least two reliable negative parasitological test results should be available to justify withholding antimalarial treatment; the optimal choice of these has yet to be identified.Paracheck had poor specificity in diagnosing malaria in severely ill children; this was likely to be due to HRP2 persistence following recent parasite clearance. The combination of positive Paracheck and negative blood slide results identified a group of children at high risk of non-typhi  The use of rapid immunochromatographic tests for malaria (RDTs) in primary care facilities has been studied over a number of years, and these tests are now being rolled out on a large scale in Africa as a means to restrict antimalarial drug use to parasitologically confirmed cases, a policy now supported by WHO . HoweverThe wider use of RDTs in patients hospitalised with severe febrile illness seems inevitable given the increasing availability of RDTs. Given current evidence of the low accuracy of routinely read slide results, this may represent an improvement over current practice  with those from double-read research slides in guiding the care of children enrolled in a 1-year study of children admitted to a district hospital for febrile illness in an area of intense malaria transmission. We compare the technical performance of Paracheck with research-quality blood slide results and suggest how HRP-2-based RDT results might contribute to clinical care in African district hospitals.Plasmodium falciparum  malaria.The study was conducted in a district hospital in north-eastern Tanzania serving a predominantly rural population with childhood mortality that is typical for Tanzania (165 deaths/1000 person years under the age of 5 years). The area is highly endemic for Details of the study have been published elsewhere  and HIV antibody tests . Blood was sent to the laboratory for full blood count  and aerobic blood culture  with identification of organisms by standard means. Blood slides were stained with Giemsa and independently double-read with discordant results resolved by a third reader. Paracheck tests were stored in a ventilated room out of direct sunlight with temperature documented not to exceed 40 °C as recommended by the manufacturer.After consenting procedures, a standard clinical history and examination based on IMCI guidelines were recorded by a study clinician . Pulse otata-10 . Final blood slide results are considered as the ‘reference standard’ result in making comparisons with Paracheck results throughout the paper.Data were scanned using Teleforms (Verity software Inc.) into MS-Access  and analysed using St-test (for parametric variable) or rank-sum (for non-parametric variable) for comparison of normally or non-normally distributed data, respectively. The logistic model was constructed with ‘positive RDT and negative blood slide’ as the dependent variable and factors in Statistical tests used chi-squared for comparison of proportions and The study was approved by the Ethics Committees of the National Institute for Medical Research, Tanzania (NIMR/HQ/R.8a/Vol.IX/392), and the London School of Hygiene and Tropical Medicine, UK (LSHTM Ethics # 2087). Witten informed consent to participate was obtained from the parent or guardian of each child in the study.P. falciparum slide positive and Paracheck positive, and 943 (25.9%) were negative to both tests. Of the 557 discordant results, 501/557 (90%) were Paracheck positive but slide negative and 56/557 (10%) were Paracheck negative and slide positive. Using the blood slide result as the reference standard, there was an inverse relationship between age-specific prevalence of parasitaemia and the specificity of Paracheck  were both aracheck .P. falciparum infections with >2000 parasites/μl with no consistent trend with increasing density above this level. The sensitivity of Paracheck was lower in detecting infections with <2000 parasites/μl and especially infections with <200 parasites/μl compared to infections with >2000/μl (The sensitivity of Paracheck was above 95% in detecting >2000/μl . HoweverP < 0.001). Similarly, bacterial infection was more common in RDT-negatives than in RDT-positives; 194/2640 (7.4%) of RDT-positive and 147/999 (14.7%) of RDT-negative children had invasive bacterial disease (P < 0.001). However, of the 501 children who were slide negative but Paracheck positive, 98 (19.6%) had invasive bacterial disease and 67 (69.1%) of these were caused by non-typhi Salmonella (NTS) infections  children in the study and these were more common in slide-negative  compared to slide-positive children . Factors judged likely to be associated with false-positive Paracheck results were assessed in the logistic regression model in Overall, 597/1850 (32.3%) of children with a true-positive Paracheck result were reported to have taken an antimalarial drug in the 2 days prior to admission compared to 188/443 (42.4%) of children with a false-positive Paracheck result  infections. False-negative RDT results at high density have been described by at least two other studies although the explanation is still not clear  or a rapidly read quality-controlled blood slide; while the latter is clearly preferable, the limitations on laboratory quality in Africa create serious challenges. More research is needed on what is sufficient evidence to justify withholding antimalarial treatment in a severely ill child admitted to hospital in malaria-endemic areas.P. falciparum infection. Thus, in areas of intense transmission of P. falciparum, patients may be overtreated with antimalarial drugs and a positive HRP2 test result should not discourage presumptive treatment with antimicrobial drugs. The combination of a positive HRP2 test with a negative blood slide result may suggest an increased risk of invasive Gram-negative septicaemia. While the Paracheck results in this study reached high levels of sensitivity, a small number of high-density P. falciparum infections were recorded as Paracheck-negative, suggesting that at least one more quality-controlled parasitological test should be used before withholding antimalarial treatment in patients with suspected severe malaria. The choice of test or combination of tests to guide treatment of children admitted to hospital with suspected malaria has not so far been defined.In conclusion, HRP-2 devices in hospitalised children in high-transmission settings suffer from low specificity that is largely dependent on the risk of recently cleared