Malaria incidence has declined considerably over the last decade. This is partly due to a scale-up of control measures but is also attributed to increasing urbanization. This study aimed to analyse the association between malaria and urbanization and the effect of urbanicity on the acquisition of semi-immunity.In 2012, children with fever presenting to St Michael’s Hospital Pramso/Ghana were recruited. The malaria-positive-fraction (MPF) of fever cases was calculated on community-level to approximate the malaria risk. The mean age of malaria cases was calculated for each community to estimate the acquisition of semi-immunity. The level of urbanicity for the communities was calculated and associations between MPF, urbanicity and immunity were modelled using linear regression.Twenty-six villages were included into the study with a mean MPF of 35 %. A linear decrease of 5 % (95 % CI: 4–6 %) in MPF with every ten-point increase in urbanicity was identified. The mean age of malaria patients increased by 2.9 months (95 % CI: 1.0–4.8) with every ten-point increase in urbanicity.The results confirm an association between an increase in urbanicity and declining malaria risk and demonstrate that the acquisition of semi-immunity is heterogeneous on a micro-epidemiological scale and is associated with urbanicity.The online version of this article (doi:10.1186/s12936-016-1138-4) contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria is the most important parasitic disease worldwide with an estimated 1.1–1.4 billion people at risk of infection .2 = 0.70) and the association between urbanicity and MPF was only slightly lower with a 4.6 %  decrease in MPF for each ten-point increase in urbanicity  with an increase of 2.9 months (95 % CI: 1.0–4.8) per ten-point increase on the urbanicity scale  in Bosumtwe District to 68 months in Chirapatre , a suburb of Kumasi. There was strong evidence for an association between urbanicity and mean age of malaria patients  with a modelled decrease of 4.9 months (95 % CI: 1.7–8.1) in the mean age per 10 % increase in MPF  in Bosumtwe District to 58 months in Gyinyase , a suburb of Kumasi. However, there was no association with urbanicity (2 0.59), economic activity , education , and housing . In the multivariate model no variable remained associated with MPF, but R2 squared increased to 0.77. The model showed the strong co-linearity between components, which was also seen in the exploratory factor analysis performed to validate the score (see Additional file The bivariate models showed that all components correlate negatively with MPF Table , while tUsing data from a hospital-based study, an ecological analysis of the association between urbanicity and clinical malaria episodes was performed. Using MPF as outcome and a continuous measure of urbanicity as exposure, a linear decrease of 5 % in MPF with every ten-point increase in urbanicity was identified. In addition, it was found that the mean age of malaria patients increased by 2.9 months with every ten-point increase on the urbanicity scale, indicating a potential influence of urbanicity on the acquisition of semi-immunity against malaria. This is supported by the finding of a lower mean age of malaria patients in communities with a higher MPF.Through the use of a continuous scale of urbanicity that provides more detailed information than a binary classification, it was possible to assess linear associations with the covariates. The vast majority of previous studies , 20, 21 The results confirm heterogeneity of malaria prevalence in areas of high endemicity as previously reported  and undeThe use of ITNs in the current study was not significantly associated with urbanicity, but surprisingly associated with a higher MPF. However, the analysis was performed on an ecological level and individual risk was not assessed. Therefore, ITN use may have been higher in communities with a high MPF as a response to higher transmission there. The reported use of ITNs was also higher than in previous studies where 22 % of children below the age of five slept under a bed net in 2006 . This diP. falciparum in an urban setting compared to a rural area [The findings of varying mean ages of malaria patients over short distances and a correlation of mean age with urbanicity and MPF indicate that the acquisition of immunity is spatially heterogeneous and possibly slower in urban areas. The association between prevalence of malaria and the acquisition of immunity is well known and has been studied on a macro-epidemiological scale and in modelling approaches , 33, 34.ral area . Howeverral area , 38 and ral area . All theral area .A difference in the age of infection between urban and rural areas and a possible delay in the acquisition of immunity has several implications for clinicians and intervention programmes. First, severe disease in urban areas may be more evenly distributed across childhood  and clinAnopheles gambiae is known to be mainly endophilic [While these findings could have major implications, the data should be interpreted with care. First, MPF is a measure of disease prevalence in patients attending the hospital and not a direct measure in the community. Calculating malaria prevalence/incidence based on data from healthcare facilities is biased by access to care and unknown hospital coverage. By using the MPF among children with fever, as previously done in several studies , 42, thedophilic . Fourth,The results demonstrate the feasibility of hospital-based studies to analyse crude associations in the micro-epidemiology of malaria and acquired immunity. However, further studies including clinical data on disease severity, especially cerebral malaria, and analyses on an individual level are needed. Future studies should also include additional risk factors such as surrounding breeding sites. Such studies will lead to a better understanding of the heterogeneity of both transmission and immunity and provide data for geographically targeted interventions.


Malaria remains one of the leading communicable diseases in Ethiopia. Early diagnosis combined with prompt treatment is one of the main strategies for malaria prevention and control. Despite its limitation, Giemsa microscopy is still considered to be the gold standard for malaria diagnosis. This study aimed to compare the performance of Giemsa microscopy with nested polymerase chain reaction (nPCR) for the diagnosis of malaria in north-west Ethiopia.Plasmodium parasites using Giemsa microscopy and standard nPCR.A cross sectional study was conducted in public health facilities in North Gondar, from March 2013 to April 2013. A total number of 297 subjects with suspected malaria were enrolled in the study. Finger-prick blood samples were collected and examined for Plasmodium falciparum and Plasmodium vivax, respectively. By nPCR, 73.1% (217/297) were malaria-positive. Among microscopy-negative samples, 13.1% (39/297) samples turned malaria-positive in nPCR. In nPCR, the rate of mixed Plasmodium infections was 4.7% (14/297) and 3.03% (9/297) were positive for Plasmodium ovale. Using nPCR as reference the sensitivity, specificity, positive predictive and negative predictive values of Giemsa microscopy were 82.0%, 93.8%, 97.3% and 65.8%, respectively, with a good agreement (κ = 0.668) to nested PCR. The sensitivity and specificity of Giemsa microscopy in identifyingAmong the study participants, 61.6% (183/297) patients tested positive for malaria by Giemsa microscopy of which, 72.1% (132/183) and 27.9% (51/183) were diagnosed as P. falciparium infections were 74.0% and 87.4% and 63.2% and 96.5% for P. vivax infections, respectively.Although Giemsa microscopy remains the gold standard for malaria diagnosis in resource-limited environments, its sensitivity and specificity as compared to nPCR is limited suggesting exploration of novel rapid and simplified molecular techniques for malaria-endemic countries. A high rate of misclassification and misidentification highlights the importance of adequate training for staff involved in malaria diagnosis. Plasmodium and continues to be one of the main causes of serious illness and death throughout the world. In spite of considerable improvements in some parts of the world, malaria-related morbidity and mortality have increased in other regions[Plasmodium to anti-malarial drugs, the distribution of mosquito vectors and immune status of the population[Malaria is a disease caused by protozoans of the genus  regions. Malariapulation.Plasmodium falciparum and Plasmodium vivax are the main species accounting for approximately 60% and 40% of malaria cases, respectively[P. falciparum to P. vivax in the country[P. falciparum is the main causative of severe malaria in Ethiopia with case fatality rates of about 10% in hospitalized adults and up to 33% in children less than 12 years old[Malaria is also among the leading communicable diseases in Ethiopia. It is estimated that 57.3 million 68%) of the population of Ethiopia live in areas at risk of malaria accounti8% of theEarly and adequate diagnosis and prompt treatment is one of the main strategies in malaria prevention, control and effective disease management. In Ethiopia, different diagnostic methods are employed but diagnosis based on clinical signs and symptoms is still most frequently used in all peripheral areas where laboratory diagnosis is not available. However, clinical diagnosis is notoriously unreliable as the sign and symptoms of malaria are non-specific and overlapping with those of other febrile diseases. In recent years the use of rapid diagnostic tests (RDTs) has gained major importance wherever laboratory diagnosis is not available . However, microscopic diagnosis of malaria based on examination of Giemsa-stained blood films remains the gold standard in Ethiopia.The sensitivity of Giemsa microscopic slide examination significantly varies across species and geographic localities. A study in Ethiopia reported a sensitivity that varied between 44% and 96%, and a specificity of greater than 90%. MicroscPlasmodium species[An earlier study from north-west Ethiopia suggested that malaria microscopy leads to 16.3% false negative, 0.7% of false positive results and the misclassification of  species. The aimThe study was conducted in a hospital  and three health centres  in North Gondar areas which are known to be endemic for malaria. The altitude ranges from 1,750 to 2100 m above sea level. According to the Municipal Health Bureau report, malaria is the most prevalent seasonal disease in these areas.A diagnostic study was conducted from March 2013 to April 2013. Male and female febrile patients of any age suspected to be positive for malaria, willing to participate and to sign the informed consent were included in this study. Patients who had received anti-malarial drugs during the past four weeks and critically ill patients unable or unwilling to provide a blood sample were excluded from the study.Plasmodium parasites were seen after examining 100 high power (1000x) fields.Study participants were recruited consecutively (convenience sampling) and a total of 297 patients with signs and symptoms consistent with malaria enrolled. Finger-prick blood samples were collected from every participant and placed on grease-free, clean microscopic glass slides. On a single slide, both thick and thin blood films were prepared. The thin blood films were fixed in methanol after air-drying before the slides were stained in a 30% Giemsa solution for 10 minutes. Thin and thick blood films were read at the health centre by experienced medical laboratory technologists and the result was considered as negative if no Two blood spots were collected from each participant, transferred on filter paper  labelled with the participant’s study code and date. Each filter paper was dried individually to avoid any chance of contamination. The samples were then stored in small plastic bags with desiccant and transported to the Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna (MUV), Vienna, Austria for molecular analysis.Plasmodium DNA from the blood spots on filter paper. Parasite detection and species classification by nested PCR assay was performed for all samples as described previously[A modified chelex-based DNA extraction method using the InstaGene Whole Blood Kit  was used for the extraction and purification of eviously,14. The Sensitivity, specificity and predictive values were determined using SISA online statistical software. The kapThe study protocol had been reviewed and approved by the institutional review board (IRB) of the University of Gondar prior to the study. Written informed consent (consent and/or assent) was obtained from all participants or their legal guardians after being translated and read in the vernacular language. Patients testing positive for malaria by microscopy received immediate treatment according to the national treatment guidelines.Plasmodium parasites in the course of the study. Among the study participants, 61.6% (183/297) patients were malaria-positive by microscopy. Of those, 72.1% (132/297) and 27.9% (51/297) were diagnosed as P. falciparum and P. vivax, respectively, by Giemsa microscopy. Nested PCR was used as reference method and 73.1% (217/297) participants tested positive by nested PCR. Among microscopy-negative patients, 13.1% (39/297) cases tested positive by nested PCR  samples were identified as mixed Plasmodium species infections by nested PCR and P. ovale was in 3.03% (9/297) patient samples  and 93.8% (95% CI: 85.4-97.7), respectively. The probability for a positive microscopy result to be true positive  was 97.3% (95% CI: 93.4-99.0) and for a negative result to be true negative  was 65.8% (95% CI: 56.2-74.3.1).Giemsa microscopy showed a good measure of agreement (κ = 0.668) with the reference method, nested PCR for P. falciparum-specific sensitivity of microscopy relative to nPCR was 74.0% (95% CI: 66.2-80.6%), the specificity 87.4% (95% CI: 80.6-92.2%), the positive and negative predictive values 86.4 (95% CI: 79.0-91.5%) and 75.8 (68.4-81.9%), respectively. The corresponding values for P. vivax were 63.2% 95% CI: (50.6-74.4%) for sensitivity, 96.5% (95% CI: 93.0-98.4%) for specificity, 84.3% (95% CI: 70.9-92.5%) for PPV, and 89.8% (95% CI: 85.2-93.2%) for NPV.Taking mixed infections into account (a mixed infection counting for both species as positive) the Plasmodium falciparum contributes around 60-75% to the malaria burden and the only other species routinely identified being P. vivax[versus fresh). In the current study, whole blood collected on filter paper has been used for nested PCR. The difference in sensitivity is obviously reflected in the different rates of positivity seen in this study (61.6% by microscopy versus 73.1% in nPCR).Malaria is still a leading cause of morbidity in Ethiopia where 78% of the country and 68% of its population are at risk of malaria.  P. vivax,17. As d P. vivax. However P. vivax. Nested  P. vivax,21 whichPlasmodium parasites. This allows for the detection of low density infections and even more importantly of mixed infections, which are routinely missed in microscopy[Nested PCR utilizes genus and species specific markers for the detection of croscopy and makeThese results are in concordance with those of other studies from Africa, Asia and Latin America reporting a considerably higher potential for detecting low parasite densities-26. A reP. falciparum was considerably higher when compared to P. vivax (74.0% vs. 63.2%), while at the same time the specificity was higher for P. vivax (87.4% vs. 96.5%) suggesting that microscopists tend to interpret slides that are difficult to read  as P. falciparum rather than P. vivax.In the present study, the sensitivity of Giemsa microscopy for the diagnosis of P. vivax-P. falciparum infections were read as malaria-positive and approximately half of them were interpreted as P. falciparum monoinfections, the other half as P. vivax. This is somewhat surprising as previous experience shows that mixed infections tend to be interpreted as P. vivax rather than P. falciparum, obviously because of its characteristic morphology.Although Giemsa microscopy did not identify a single case of mixed infection, all P. falciparum and P. vivax had not been reported from the region[P. ovale out of which six were monoinfections and three were mixed with P. falciparum. However, only three were interpreted as being negative by microscopy. Once again not a single sample tested positive for Plasmodium malariae.Until very recently, species other than e region. This isIn spite of the inherent limitations of malaria Giemsa microscopy, the quality of microscopic diagnosis largely depends on the quality of training. On the The added cost of using molecular techniques as confirmatory diagnostic tool for the malaria control programs needs to be carefully balanced against the cost (not to mention the human cost) of a higher disease burden. In the near future PCR  will not be available throughout most malaria-endemic regions but a network of reference centers could potentially support ongoing diagnostic and control efforts by malaria control programs on the long run even significantly reduce the overall cost by providing a more targeted approach to the challenges of the malaria eradication era.The authors declare that they have no competing interests.AA and HN conceived the study. AA and HFP undertook statistical analysis, undertook molecular analysis of the samples and drafted the manuscript. GG, SG and AK contributed in proposal writing, blood sample collection and microscopic diagnosis of malaria slides. All authors contributed to the writing of the manuscript and approved the submitted version of the manuscript.


Plasmodium falciparum prevalence and incidence.Mass drug administration (MDA) and focal MDA (fMDA) using dihydroartemisinin plus piperaquine (DHAp), represent two strategies to maximize the use of existing information to achieve greater clearance of human infection and reduce the parasite reservoir, and provide longer chemoprophylactic protection against new infections. The primary aim of this study is to quantify the relative effectiveness of MDA and fMDA with DHAp against no mass treatment (standard of care) for reducing The study will be conducted along Lake Kariba in Southern Province, Zambia; an area of low to moderate malaria transmission and high coverage of vector control. A community randomized controlled trial (CRCT) of 60 health facility catchment areas (HFCAs) will be used to evaluate the impact of two rounds of MDA and fMDA interventions, relative to a control of no mass treatment, stratified by high and low transmission. Community residents in MDA HFCAs will be treated with DHAp at the end of the dry season (round one: November to December 2014) and the beginning of the rainy season (round two: February to March 2015). Community residents in fMDA HFCAs will be tested during the same two rounds for malaria parasites with a rapid diagnostic test; all positive individuals and all individuals living in their household will be treated with DHAp. Primary outcomes include malaria parasite prevalence , as measured by pre- and post-surveys, and malaria parasite infection incidence , as measured by a monthly longitudinal cohort. The study is powered to detect approximately a 50 % relative reduction in these outcomes between each intervention group versus the control.Strengths of this trial include: a robust study design (CRCT); cross-sectional parasite surveys as well as a longitudinal cohort; and stratification of high and low transmission areas. Primary limitations include: statistical power to detect only a 50 % reduction in primary outcomes within high and low transmission strata; potential for contamination; and potential for misclassification of exposure.NCT02329301. Registration date: 30 December 2014.Identifier: Clinicaltrials.gov: The online version of this article (doi:10.1186/s13063-015-0862-3) contains supplementary material, which is available to authorized users. The recent Zambian National Malaria Strategic Plan 2011 to 2016 calls for ambitious efforts to work towards malaria elimination, with the establishment of at least five malaria-free zones by 2015 [Plasmodium falciparum parasite, thereby preventing onward transmission from infected individuals.During the 2012 dry season (June to November) in Southern Zambia, the effectiveness of three large-scale malaria mass test and treatment (MTAT) intervention rounds were assessed with a community randomized controlled trial (CRCT) . The MTAP. falciparum [The results of the Zambia MTAT trial , as welllciparum . Shiftinlciparum , 8.The results from the previous MTAT trials also suggest that alternatives to the MTAT antimalarial delivery strategy are needed. One alternative strategy to avoid some important limitations of MTAT involves treating all individuals within a specified target area with an effective antimalarial, irrespective of having a parasite infection identified by an RDT, which would constitute community-wide mass drug administration (MDA) . There aP. falciparum infections identified by RDTs at the household level, suggesting that if one person tests positive, there is a higher chance (than random) that others in the house are also infected, potentially including those with low-density infections missed by RDTs. Such spatial clustering of infections in other settings has been well documented [An alternative to community-wide MDA is focal MDA (fMDA), which combines aspects of both MTAT and MDA. fMDA, in this trial, consists of testing all individuals in the community for parasites using RDTs and treating those that are positive. But in households where anyone had a positive RDT, all household members are treated with an effective antimalarial, such as DHAp (MDA-DHAp), irrespective of each person’s RDT result. As such, fMDA uses real-time information from RDT results to focus MDA activities to populations at greatest risk of having, as well and perhaps acquiring, an infection. Results of the 2012 MTAT-AL data analysis in this study site of Southern Province shows considerable clustering of cumented . FMDA-DHP. falciparum parasite prevalence, confirmed out-patient malaria case incidence, and monthly infection incidence in areas stratified by high and low malaria transmission. It is hypothesized that two rounds of fMDA and MDA with DHAp will be significantly more effective than no mass treatment (standard of care) at reducing these health outcomes. A secondary objective of this study is to compare the cost and cost-effectiveness of the fMDA and MDA strategies with DHAp compared to no mass treatment.MDA and fMDA using DHAp represent two potential strategies to achieve greater clearance of human infection and reduction in the parasite reservoir, and both provide longer chemoprophylactic protection against new infections. The primary aim of this study is to quantify the relative effectiveness of MDA and fMDA with DHAp against no mass treatment for reducing Other outcomes related to the quality of program implementation will also be measured during this study to help understand why the MDA and fMDA with DHAp interventions were, or were not, found to be effective in this setting. These outcomes include: participant adherence of taking a full course of DHAp by the fMDA and MDA interventions; clearance of asexual stage parasites at day seven following the administration of DHAp; population coverage of the fMDA and MDA interventions in the study areas; and community acceptability of participating in the fMDA and MDA interventions. Measurement for these outcomes is summarized in the Methods/Design section below, with further details provided in the Additional file The study will be conducted in Southern Province Zambia in districts along Lake Kariba, including Gwembe, southern Kalomo, Siavonga, and Sinazongwe Fig. . The popThe area is primarily composed of the Tonga ethnic group. Livelihood typically consists of fishing and farming. While there are towns in the study area, the majority of the area is rural. The health system throughout the study area consists primarily of public sector health facilities, with health facility catchment areas (HFCAs) typically covering 500 to 1,500 households. All HFCAs were mapped and geographically defined prior to this study.Malaria transmission in this area is low to moderate, with malaria parasite prevalence ranging from less than 1 % in some areas to more than 25 % in other areas. The high malaria transmission season in the study area follows the rainy season and lasts from January to May. Results from the 2012 MTAT-AL intervention identified two distinct areas of malaria transmission intensity, with higher transmission areas along the low-lying shores of Lake Kariba, and lower transmission areas inland from Lake Kariba.There are current efforts to maintain high vector control coverage in the study area, with household coverage of LLINs estimated to be between 60 and 80 %. Additionally, a comprehensive IRS campaign to cover all households in the study area was conducted between November 2014 and January 2015, using the highly effective insecticide Actellic.P. falciparum parasite infections. DHAp was recently approved as the alternate first-line treatment in Zambia for uncomplicated malaria [DHAp is the study drug used in this trial to treat  malaria . The DHAAll individuals meeting the inclusion and exclusion criteria to receive DHAp will be offered an age-appropriate course of the drug, according to the manufacturer’s recommendations and the national guidelines. DHAp dosing will be age-based  and February 2015 (beginning of rainy season) in selected study site HFCAs. Spacing of the rounds was determined by the manufacturer recommendation of not receiving more than two courses of DHAp within two months [Under coordination with the Zambia NMCC, two rounds of fMDA with DHAp are being implemented in December 2014 (end of dry season) and February 2015 (beginning of rainy season) in selected study site HFCAs. Each round of the fMDA-DHAp intervention consists of testing all individuals with an RDT and then sharing the result of the RDT. An age-appropriate therapeutic dose of DHAp is offered to all consenting individuals meeting the inclusion criteria to receive DHAp, as outlined below and in Fig. A modified directly observed treatment (DOT) will be used to maximize adherence with the prescribed DHAp treatment among all individuals participating in the MDA intervention and all individuals in households with a member with a positive RDT in the fMDA intervention. At the community level, DHAP will be given by DOT on the first day of the household visit to all household members by the campaign team testing pairs. The second day of the treatment will not be given by DOT by campaign teams, but will be asked on the final day of treatment by an adherence monitoring officer working with the campaign teams. These adherence officers will make the second household visit to administer DOT for the third treatment dose, ask about adherence for the second dose, observe and count the treatment packets for the remaining pills, and provide additional instruction on full compliance if needed.Figures Pregnancy status during the first trimester will be determined among women ages 12 to 49 years through direct questioning, and by offering a urine human chorionic gonadotropin rapid pregnancy test at the field visit for women unsure of their pregnancy status or reporting longer than five weeks since last menstruation. All consenting women in the MDA and fMDA interventions with a negative RDT deemed to be in the first trimester of pregnancy will receive no malaria treatment. All consenting women deemed to be in their first trimester of pregnancy with a positive RDT will receive the recommended standard of care treatment for uncomplicated malaria in Zambia among women in their first trimester of pregnancy, which consists of age-appropriate treatment with oral quinine for seven days.All individuals with suspected severe malaria or other severe illness  urine) will be referred to the nearest health facility for treatment, and will not be included in the MDA or fMDA interventions or associated research.MDA requires high levels of participation and adherence to the full treatment course to be as effective as possible. To encourage participation, several activities were conducted in advance of and during the treatment campaigns in order to broaden and strengthen community awareness. First, information about the campaigns, the treatments, and the process were shared through the Ministry of Health and Ministry of Community Development, Mother and Child Health structures, including among community health workers to begin discussions at local levels. Second, community sensitization meetings were held throughout campaign areas across nine districts to inform leaders at the district level, including district councilors, religious leaders, and lead educators, and at the chiefdom level, with special outreach to chiefs, village headmen, local community health workers, and community members gathered for village entry meetings. Information, education, and communication materials were developed with the Zambia NMCC on malaria and pretested in campaign areas, containing behavior change communication messages for community members and community leaders. These were translated into the local language and distributed at these community sensitization meetings. Third, radio spots were developed and aired in the local language in target areas, along with a series of radio group episodes in which community members engaged with campaign health experts to learn about the program and discuss the benefits of participation.Standard of care in Zambia, including the study area in Southern Province, consists of identification of malaria infections and clinical cases through passive case detection at local health facilities or community health workers. Zambian national policy stipulates that individuals seeking care for a fever be given a malaria diagnostic test by RDT or microscopy. Individuals with confirmed malaria are then treated with the first-line antimalarial in Zambia, which is AL. When no diagnostic test is available at a health facility, a clinical diagnosis is made based on signs and symptoms, and treatment follows the identical protocol as with RDT-confirmed malaria cases. Standard of care does not include any kind of active surveillance for malaria parasite infection or mass treatment of the population with antimalarials.A CRCT will be used to evaluate the impact of two rounds of the MDA-DHAp or fMDA-DHAp (the two intervention arms) at reducing malaria parasite infection prevalence and incidence, as well as malaria-related morbidity, compared to a control group receiving standard of care. Communities, or clusters, are defined by HFCAs and serve the unit of randomization. A total of 60 health facilities and their catchment areas are included in the study.As the impact of the MDA and fMDA interventions are expected to vary across malaria transmission strata, the 60 HFCAs included in the study have been stratified into high and low transmission strata. To do this, parasite prevalence in each HFCA was estimated with a kriged continuous surface of parasite prevalence from data from the baseline 2014 parasite survey. Catchments were then ranked from highest to lowest parasite prevalence. The top 30 HFCAs were included in the high transmission strata, while the bottom 30 were included in the low transmission strata; the resulting cut point between the high and low transmission strata was approximately 10 % parasite infection prevalence.Pre-existing maps of HFCAs were used to classify households as belonging to a specific HFCA, which serve as the unit of randomization. To ensure balance across large and small HFCAs, which could potentially affect the quality of program implementation and other related unobserved confounding factors, HFCAs were characterized as having either a large or small population catchment size. This classification was based on a cut-point obtained from the mean population catchment size across the 60 HFCAs. Within the high and low transmission strata and the large and small population catchment size classifications, the random allocation rule will be used to assign HFCAs to the fMDA-DHAp, MDA-DHAp, or control group. As a result of this process and the stratification outlined above, the 60 total HFCAs are split into 10 HFCAs per treatment group  in the high transmission strata (n = 30 HFCAs), and 10 HFCAs per treatment group  in the low transmission strata (n = 30 HFCAs).After allocation, the appropriate intervention will be implemented in the entire catchment area according to treatment group assignment. Allocation of study arms will not be blinded to the deliverers of the intervention or to the main investigators, as delivery of the intervention precludes the possibility of allocation concealment. However, data collectors in the follow-up parasite prevalence survey, as outlined below, will be blinded to allocation. All primary analyses will also be blinded to analysts by the use of concealed code for the study arm variables in the final dataset.P. falciparum infection (based on both RDT and PCR) out of all children < six-years-old tested within the 2014 and 2015 cross-sectional parasite surveys.Parasite prevalence during the high transmission season among children < six-years-old: defined as the proportion of children < six-years-old (≥three months to < six-years-old) with P. falciparum infection incidence rate among individuals ≥ three-months-old: defined as the number of individuals ≥ three-months-old with a new P. falciparum infection (based on PCR) divided by the total person-time observed among a cohort of individuals ≥ three-months-old followed up on for a 12-month-period.Total and confirmed outpatient (OPD) malaria case incidence and inpatient (IPD) malaria case incidence among all ages: defined as the number of OPD and IPD malaria confirmed and suspected cases per person per year, as ascertained from the routine rapid reporting system; facility catchment population size estimates will be used for the exposure denominator.RDT positivity: defined as the proportion of all individuals tested by RDT at each round of the fMDA and MDA interventions (and among a contemporaneous sample in the control group), with a positive RDT, among the population of individuals ≥ three-months-old.Primary outcomes used to measures the impact of the MDA-DHAp and fMDA-DHAp interventions, as compared to a control of standard of care, are:For all data collection procedures, written informed consent will be obtained from each participant prior to study enrollment in the trial.P. falciparum parasite infections will be measured using RDTs, microscopy, and PCR, depending on the study outcome, as described below. The RDTs used in this trial are manufactured by  under the brand name SD Bioline Malaria Antigen P.f, which detects the Histidine-rich protein 2 (HRP-2) antigen of P. falciparum. This RDT is approved by the WHO, Global Fund, and the Zambian Ministry of Health. RDTs are used across all data collection methods in this trial, including the parasite surveys, longitudinal cohort, fMDA and MDA rounds, and at health facilities in the study area. Detection of malaria parasite infections from microscopy is based on thick smears. Thick blood smears for microscopy will be used in the parasite surveys and the longitudinal cohort. PCR will be performed from capillary blood collected on dried blood spots (DBS) on filter paper, to identify low-density asexual and sexual stage parasite infections, as part of the parasite surveys and the longitudinal cohort.A cross-sectional survey of households in the high transmission season (April to May) in 2014, and again in 2015, will be conducted to obtain an unbiased sample of children under six-years-old (excluding those under one-month-old) for measuring primary outcome one . All children under six-years-old that slept in the house the night before the survey will be included in the sample. A complete enumeration and geo-referencing of all households in the study area serves as the sampling frame. To mitigate contamination between exposure groups  in the household sampling frame, sampling buffers of at least 1.5 km were derived for all HFCAs using GIS, in order to maintain an adequate distance between households in HFCAs with different exposure assignments (resulting in a gap of at least 3 km between sampled households in different HFCAs). A simple random sample of 47 households was then drawn within each of the 60 HFCAs , to obtain the total sample size of 2,820 households in the 2014 baseline survey, after restricting the sampling frame for sampling buffers in GIS. The same sampling procedure will be used for the follow-up parasite survey in 2015.Selected households during each survey round will be visited by trained survey data collectors that will administer a standardized survey questionnaire using mobile phones, based on the standard Malaria Indicator Survey (MIS) questionnaire . CapillaP. falciparum infection incidence will be measured by following a cohort of individuals aged three-months-old and older (and not pregnant) for measuring primary outcome indicator two, the P. falciparum infection incidence rate  rapid reporting system, from all 60 health facilities in the study area. Data from this source are accessible via a web server with password protection. This system was established in 2011 and 2012 and has been running in the catchment areas ever since. Time series data by facility week will be collated from this system to provide a passive incidence-based data set for evaluation of the impact of the MDA and fMDA interventions, as compared to a control of no mass treatment, on primary outcome three. Confirmed and total case counts will be standardized by the estimated mid-year populations of each HFCA.As described above under the MDA and fMDA intervention descriptions, all individuals aged three-months-old and older in these HFCAs will be tested by RDT for detecting malaria parasite infections; this will then be used for ascertaining primary outcome four (RDT positivity) in each HFCA. RDT positivity in control areas during each of the two rounds of the MDA and fMDA interventions in 2014, and again in 2015, will be obtained through comparing RDT positivity among individuals tested as part of the monthly control cohort during the months when the MDA and fMDA rounds occur.Cost data collection will be coupled with the study in order to provide numerator estimates for cost-effectiveness calculations. Cost data collection will be provider focused and will not require collection of data from intervention participants or research subjects. Data on the cost of inputs to the intervention will be collected using a review of program records and reports, invoices, budgets, expenditure reports, and interviews with intervention implementers. Interviews will be focused on resource use during the implementation of the study interventions. Where direct estimates of unit costs for inputs are not available through the above methods, data on costs will be supplemented with secondary source data, such as is available from the WHO-CHOICE database, the National Bank of Zambia, the World Bank, the International Monetary Fund, or other published literature. Economic costs will be estimated, meaning that costs from donated inputs will also be valued.The maximum number of cluster units available was 60, split evenly with 10 clusters per MDA, fMDA, and control groups in the high and low transmission strata. Sample size was derived using a formula for CRCTs developed by Hayes and Moulton . Sample The coefficient of variation (CV) was estimated using data from a previous study of the 2012 MTAT-AL in the study area (CV = 0.31). Based on the number of clusters available in the high transmission strata , a statistical power of 80 %, a probability of committing a type two error of 5 % (one-tailed test), and simple random sampling used to obtain the sample of children in each household survey, a sample size of 39 children under-years-old per HFCA per treatment group will be needed to detect 12 % absolute detectable difference in parasite prevalence between any two groups, from a baseline of 30 % . This detectable difference equates to a crude odds ratio of 0.51. With survey non-response assumed to be 20 %, a sample size of 470 children per treatment group (47 children per cluster), totaling 1,410 children under six-years-old per survey round will be sought , the observed CV (CV = 0.41), a statistical power of 80 %, a probability of committing a type two error of 5 % (one-tailed test), and simple random sampling used to obtain the sample of children in each household survey, a sample size of 39 children under six-years-old per HFCA per treatment group will be needed to detect 6 % absolute detectable difference in parasite prevalence between any two groups from a baseline of 10 % . This detectable difference equates to a crude odds ratio of 0.38. With survey non-response assumed to 20 %, a sample size of 470 children per treatment group (47 children per cluster), totaling 1,410 children under six-years-old per survey round will be sought . Based on the 30 HFCAs available in the high transmission strata, a statistical power of 80 %, a probability of committing a type two error of 5 % (one-tailed test), a sample size of 25 individuals aged three-months-old and older per HFCA (cluster) per treatment group will be needed to detect an absolute detectable difference in 12-month cumulative parasite infection incidence between any two groups from an assumed baseline of 1.8 infections per person per year to 0.91 infections per person per year (a 49 % relative reduction). With a loss to follow-up rate assumed to be 33 %, a sample size of 375 individuals per treatment group (38 per HFCA) will be enrolled and followed up on monthly, totaling 1,125 individuals aged three-months-old and older enrolled in the cohort study in the high transmission strata . Based on the 30 HFCAs available in the low transmission strata, a statistical power of 80 %, a probability of committing a type two error of 5 % (one-tailed test), a sample size of 27 individuals aged three-months-old and older per HFCA (cluster) per treatment group will be needed to detect absolute detectable difference in parasite infection incidence between any two groups from an assumed baseline of 0.6 infections per person per year to 0.28 infections per person per year (a 53 % relative reduction). With a loss to follow-up rate assumed to be approximately 33 %, a sample size of 375 individuals per treatment group (38 per cluster) will be enrolled and followed up on monthly, totaling 1,125 individuals enrolled in the cohort study in the low transmission strata  will be achieved. As described above, a sample size of 750 individuals aged three-months-old and older in the cohort in the control HFCAs will provide contemporaneous measure of RDT positivity in areas without intervention rounds . This sample size in areas of high transmission will provide 80 % statistical power to detect a 33 % difference  in RDT positivity between the control group and either of the MDA groups, assuming a baseline prevalence of 30 %, a CV = 0.31, 10 HFCAs in each treatment group, and a probability of committing a type two error of 5 % (one-tailed test). This sample size in areas of low transmission will provide 80 % statistical power to detect a 50 % difference  in RDT positivity between the control group and either of the MDA groups, assuming a baseline prevalence of 10 %, a CV = 0.41, 10 HFCAs in each treatment group, and a probability of committing a type two error of 5 % (one-tailed test).The primary analysis for estimating the treatment effect between MDA, fMDA, and control HFCAs for primary outcome one  and two (parasite infection incidence rates) will be based on intention to treat at the individual level for infections measured by RDT and PCR. The analysis of parasite prevalence will use a logistic or log binomial model, while the analysis for infection incidence will use a Poisson or negative binomial model. Models will include a random intercept at the HFCA level (randomization unit), fixed effect for intervention exposure, time period (pre- and post-intervention), plus other potential confounding factors. Standard errors will be estimated, with the addition of repeated measures among individuals.Data for total and confirmed OPD and IPD malaria case incidence (outcome three) among all ages will be analyzed on an intention-to-treat basis. Routine data on case counts will be analyzed using a Poisson or negative binomial model, with random intercepts at the HFCA level. The models will include a fixed effect for study arm and time period (pre- and post-intervention), plus other potential confounding factors.X2 test, as well as logistic regression models to account for potential confounding factors, on an intention-to-treat basis. Logistic models will include a random intercept for HFCA, as well as fixed effects for other potential confounding factors. Trends in parasite prevalence across rounds, with the inclusion of the control group, will then allow rapid assessment of the effect of the fMDA and MDA interventions over time, after removing the seasonality effect.Differences in prevalence measures (RDT positivity) at each round will be assessed using an Standard methods for collecting cost data for the specific components of the work will be used to then summarize intervention costs. Cost-effectiveness calculations will be undertaken utilizing cost data collected as above, as well as the findings of the study evaluation on the effectiveness of the intervention. The primary cost-effectiveness outcome will be the calculation of the incremental cost effectiveness ratios (ICERs) comparing the cost-effectiveness of MDA to the standard of care, and the ICER of fMDA alone versus standard of care. Secondary outcomes will include the ICER of MDA versus fMDA alone, and the stratified analysis of all three previously listed outcomes by high and low prevalence settings. Further details of the cost and cost-effectiveness analysis are presented in the Additional file The population coverage of the MDA and fMDA interventions will be measured in two ways at each intervention round. Operational program coverage is defined as the proportion of individuals aged three-months-old and older, and households visited, offered the MDA and fMDA interventions within the target areas. Effective program coverage is defined as the proportion of individuals (aged three-months and older) that agreed to participate in the MDA and fMDA interventions among all individuals eligible to participate in the intervention in the target population.The operational coverage will be estimated as the percent of the population that received a visit from the mass treatment teams to offer the MDA or fMDA interventions, among those eligible for inclusion. This will be obtained from a combination of MDA and fMDA program data and census enumeration data. Additionally, the proportion of individuals accepting the MDA and fMDA interventions aged three-months-old and older, among those eligible for inclusion in the intervention, will provide an estimate of the effective coverage of each program, as outlined below. Data for the denominator of individuals and households targeted for the intervention will be ascertained from the household enumeration for the sampling frame. Individual, household, and community level factors associated with coverage will be assessed using mixed effects logistic regression.A total of 150 individuals enrolled in the cohort study at round one, with a positive RDT in the MDA (n = 75) and fMDA (n = 75) rounds, plus another 150 individuals enrolled in the cohort study at round one, with a negative RDT in the MDA round, will be used to assess the level of adherence with the prescribed DHAp antimalarial regimen. Further details of the primary outcomes, sample size calculations, data collection methods, and the statistical analysis plan for this sub-study are provided in the Additional file A total of 150 individuals enrolled in the cohort study at round one, with a positive RDT in the MDA (n = 75) and fMDA (n = 75) rounds, will be used to assess parasite clearance following DHAp. Further details of the primary outcomes, sample size calculations, data collection methods, and the statistical analysis plan for this sub-study are provided in the Additional file The assessment of community acceptability of the MDA and fMDA interventions will be ascertained using a mixed methods approach. Acceptability of the interventions will be assessed quantitatively during the baseline and follow-up parasite surveys among WRA. Focus groups will also be used to qualitatively assess community perceptions of the acceptability of the MDA and fMDA interventions, including community members who consented and participated, as well as those who refused. Further details of the acceptability study are provided in the Additional file Figure Ethical approval in Zambia for the primary and secondary studies has been obtained from the Research Ethics Committee (REC) at the University of Zambia (study reference 007-03-14) and the Zambian Medicines Regulatory Authority . Ethical approval was also obtained from the Tulane Institutional Review Board  and Western IRB .In line with the goals of the Zambia National Malaria Strategic Plan, robust comparisons of the effectiveness and cost-effectiveness of available mass treatment approaches, including MDA and fMDA interventions using DHAp, are needed. Toward that end, this study will assess the relative effectiveness of two mass treatment approaches at reducing the malaria burden beyond already implemented interventions of LLINs and IRS. The relative effectiveness of MDA and fMDA with DHAp will be established against a comparison of no mass treatment (standard of care) in areas with higher and lower malaria parasite prevalence using a community randomized control trial. The findings of this study will provide the Zambia NMCC and their partners with guidance on the most effective and cost-effective strategies to further reduce the size of the malaria parasite reservoir, malaria transmission, and the incidence of clinical disease in southern Zambia, and will hopefully show evidence of local transmission interruption.The strengths of this trial include the fact that a rigorous study design (a CRCT) will be used to assess the impact of the MDA and fMDA interventions, as compared to a control of no mass treatment (standard of care). The study is also powered to assess differences between each treatment group (MDA and fMDA) and the control group, within areas of high and low transmission, with 80 % statistical power. The study uses robust measures of malaria parasite infection at the point of contact using RDTs, as well as PCR, for detecting low-density parasite infections, as part of the parasite surveys and the longitudinal cohort. The study also uses multiple methods for measuring malaria-related outcomes, including malaria parasite infection prevalence ascertained from a pre- and post-parasite survey, malaria parasite infection incidence using a longitudinal cohort of individuals selected randomly from within each HFCA, and confirmed malaria case incidence throughout the study using routine health management information system (HMIS) data in the study area. Buffer areas between contiguous HFCAs with different interventions are being used to limit contamination and misclassification bias. Measures of the quality of the implementation of the MDA and fMDA interventions will be obtained to help with the interpretation of results; these include MDA and fMDA population coverage, adherence to DHAp, parasite clearance after DHAp, community perceptions and acceptability of the MDA and fMDA interventions, and multiple measures of operational coverage. Lastly, a rigorous analysis plan will be followed, for assessing the impact of each intervention compared to a control group, while accounting for potential contextual and confounding factors within random-effect regression models.Noted weaknesses of this trial include a less than ideal number of community randomization units (N = 60), especially when stratified by high (n = 30) and low (n = 30) transmission strata. The sample size within the high and low transmission strata will only allow the detection of approximately a 50 % reduction in parasite prevalence and infection incidence between intervention groups, with 80 % statistical power; the threat of a type two error is therefore higher than ideal. The primary analysis will be based on the intention-to-treat principle; there is potential for misclassification of exposure using this approach. Secondary analysis using a per-protocol approach will be used mitigate any misclassification of exposure. And lastly, there is always the potential for error in the measurement of outcomes, exposure to the interventions, and potential confounding factors in such a large and complex trial. We have gone to considerable length to try and minimize such error through the use of electronic data capture systems in the field and robust diagnostic methods.The study is ongoing. The baseline parasite survey was completed in April to May 2014. The first round of the MDA and fMDA intervention was completed in November to December 2014; the longitudinal cohort started at the same time. The second round of the MDA and fMDA intervention is scheduled for February 2015 (concluding in mid-March 2015). The follow-up parasite survey is scheduled for April to May 2015, while monthly longitudinal follow-up is scheduled to continue through November 2015.


Glucose-6-phosphate dehydrogenase (G6PD) deficiency and hemoglobinopathies were the inherited conditions found mostly in African. However, few epidemiological data of these disorders was reported in Equatorial Guinea (EQG). This study aimed to assess the prevalence and healthy effects of G6PD deficiency and hemoglobinopathies among the people on malaria endemic Bioko Island, EQG.Blood samples from 4,144 unrelated subjects were analyzed for G6PD deficieny by fluorescence spot test (FST), high-resolution melting assay and PCR-DNA sequencing. In addition, 1,186 samples were randomly selected from the 4,144 subjects for detection of hemoglobin S (HbS), HbC, and α-thalassemia deletion by complete blood count, PCR-DNA sequencing and reverse dot blot (RDB).- (G202A/A376G) were detected in 356 cases (99.2%), G6PD Betica (T968C/A376G) in 3 cases. Among the 1,186 subjects, 201 cases were HbS heterozygotes, 35 cases were HbC heterozygotes, and 2 cases were HbCS double heterozygotes; 452 cases showed heterozygous α-thalassemia 3.7 kb deletion (-α3.7 kb deletion) and 85 homozygous - α3.7 kb deletion. The overall allele frequencies were HbS 17.1% (203/1186); HbC, 3.1% (37/1186); and –α3.7 kb deletion 52.4% (622/1186), respectively.The prevalence of malaria and anemia was 12.6%  and 32.8% , respectively. Overall, 8.7% subjects  were G6PD-deficient by FST, including 9.0%  males and 7.9%  females. Among the 359 G6PD-deficient individuals molecularly studied, the G6PD AHigh G6PD deficiency in this population indicate that diagnosis and management of G6PD deficiency is necessary on Bioko Island. Obligatory newborn screening, prenatal screening and counseling for these genetic disorders, especially HbS, are needed on the island. Glucose-6-phosphate dehydrogenase (G6PD) deficiency and hemoglobinopathies are widespread human erythrocyte genetic diseases affecting millions of people ,2. The g- variant (G202A/A376G) predominates in sub-Saharan Africa where it affects 15 to 20% of the African population [Plasmodium vivax and Plasmodium ovale malaria to prevent relapses, and as a single-dose or short-course gametocytocidal drug with the goal of reducing transmission of falciparum malaria. Despite the therapeutic advantages of PQ, the wider use of the drug is restricted by their toxicity profile. The most important adverse effect of PQ is dose-related hemolysis, which could potentially create significant morbidity and undermine confidence in PQ prescription [P. falciparum transmission and achieving the radical cure of Plasmodium vivax.G6PD deficiency is prevalent in Africa, the Middle East, the Mediterranean and Southeast Asia . Among t G202A/A36G predomcription . TherefoGAG>GTG mutation at codon 20 of β-globin gene. Heterozygote individuals (HbAS) are generally asymptomatic, and homozygote individuals (HbSS) have lifelong acute and chronic complications [+-thalassemia is the other common hemoglobinopathy in Africa [+-thalassemia causes slight hematological changes and homozygote carriers show mild microcytic anemia. Although these genetic disorders are a major public health problem and social burden for people in endemic regions, few data are available about the genetic frequencies of these disorders in some sub-Sahara regions.Anemia in children continues to be a major public health challenge in most developing countries, particularly Africa . Althougications ,14. α+-t2. It is divided into a mainland territory, which is bordered by Cameroon to the north and Gabon to the east and south, and five small islands, including Bioko, Corisco, Annobón, Elobey Chico and Elobey Grande. Although innovative malaria control programs in EQG have had a marked impact on malaria infection and mortality, malaria due to Plasmodium falciparum is still the major public health problem in the country. The World Health Organization (WHO) estimates that the probability of dying among children under 5 years of age in EQG is 205 of 1,000 live births, with 24% of death attributable to malaria [Equatorial Guinea (EQG) is a country located in central Africa, with an area of 28,068 km malaria . BecauseHere, we performed a large-scale molecular epidemiological survey of G6PD deficiency and hemoglobinopathies in 4,144 subjects, which firstly allows a sight into the prevalence and molecular characterization of these genetic diseases on Bioko Island. It is a prerequisite for defining a specific policy for these genetic diseases screening, genetic counseling, prenatal diagnosis and implementation of PQ for malaria on the island.Plasmodium falciparum is still the major public health problem on the island. The entomological inoculation rates (EIR) on Bioko Island ranged from 163 to 840, with the outdoor EIRs reaching > 900 infective mosquito bites per year [Bioko Island belongs to EQG and is located in the Gulf of Guinea, about 100 km off the coast of southern Nigeria and 160 km northwest of continental EQG . The islper year .The study population included 4,144 unrelated subjects living on Bioko Island These subjects aged from 1 to 75 years and received the screening for G6PD deficiency and hemoglobinopathies in Malabo Regional Hospital during April 2012 and May 2014. Questionnaires about nationality, gender, age, native status or not and written consent forms were available in Spanish (the most-common language) to ensure comprehensive understanding of the study objectives. This study was approved by the ethics committees of Malabo Regional Hospital and the First Affiliated Hospital of Shantou University Medical College. All subjects or their parents gave their informed consent by signature or thumbprint.2 anti-coagulated tubes from each subject according to the standard procedures described previously [Plasmodium falciparum cassette test  and positive samples were treated with artesunate-amodiaquine (Camoquin plus).Blood samples were collected with EDTA-Keviously , and 200n = 4,144) were detcted for G6PD deficiency by a commercial fluorescence spot test (FST) kit  as previous report [All dried blood spots . The DNA concentration was measured by UV-2000 spectrophotometry , then adjusted to 40 ng/μL for PCR. The molecular analysis strategies for screening G6PD mutations were shown in G>A) and A376G (c.376 A>G) mutations by high-resolution melting (HRM) assay as described previously [All G6PD-deficient samples were analyzed for G202A . Normalized melting curves showed the fluorescence signal against temperature, and derivative plots showed melting temperature peaks. The pre-identified DNA including G202A, A376G and wild-type DNA, were used as standard references. When the plots of samples were classified by the standard reference, they were identified as the same genotype of the standard. 12 exons of G6PD gene were amplified and sequenced in G6PD-deficient samples without these two mutations, the primers and conditions were described previously .A total of 1,186 adults were randomly selected from the 4,144 subjects for hematological analysis with the conditions as follows: (1) individuals >16 years old; (2) not pregnant women. Hematological data were collected by an automated blood counter . Anemia was defined as hemoglobin level <13.0 g/dL for males and <12.0 g/dL for females. The cut-off values for anemia were >6.0 g/dL, severe; 5.9–9.0 g/dL, moderate; and <9.0 g/dL, mild.Genomic DNA was extracted by a DNA blood mini kit . DNA concentration was mesured by UV-2000 spectrophotometry . Then DNA sample concentration was adjusted to 40 ng/μL for PCR.A>T) and HbC (c.19 G>A) were analyzed in the 1,186 subjects by PCR-DNA sequencing. PCR was amplified with Taq polymerase  in a MJ Mini Personal Thermal Cycler (Bio-RAD) as described previously [5’-AAGGCTGGATTATTCTGAGTC-3’, and P2, 5’-CACTTGCCCGAGTCTGTTT-3’. Reactions involved initial denaturation at 95°C for 3 min, 35 cycles of PCR . DNA sequencing was performed by the ABI 3730xL DNA Sequencer .HbS , the 1,186 samples were analyzed for 3 known α-thalassemia deletions  by a commercial thalassemia reverse dot blot (RDB) gene chip  [Because most cases of -α, China) ,23. The P<0.05 was considered statistically significant.Statistical analysis involved use of SPSS 16.0 . The prevalence of α-thalassemia alleles was calculated from the Hardy-Weinberg formula. Data for the 3 regions on Bioko Island and previous studies were analyzed by chi-square test. Plasmodium falciparum malarial parasite was present in 12.6% cases . Amongst these samples, 8.7%  participants were G6PD deficient. The prevalence of males and females were analyzed separately. G6PD deficiency was found in 9.0%  of males and 7.9%  of females (p = 0.238). Because of the randomly inactivation of X-chromosome in female, many heterozygous females were expected to have a normal phenotype. These heterozygous females with a normal phenotype could not be detected by FST. Therefore, the detection rate of G6PD deficiency in males was little higher than in females.4,144 subjects, including 2,758 males (66.6%) and 1,386 females (33.4%), received screening for G6PD deficiency. The mean age was 30 year (SD 28.3). – variant (G202A/A376G) by HRM assay. Melting curves for G202A and A376G mutations were shown in Of the 359 G6PD-deficient samples, 356  were detected with G6PD An = 18), 5.7% moderate (n = 67) and 25.6% mild (n = 304). The burden of anemia was higher for females than males  (p = 0.010).Among the 1,186 adults that underwent detection of hemoglobinopathies, the prevalence of anemia was 32.8% : 1.5% severe , which accounted for 52.4%  of allele frequencies and was present in a heterozygous state, −α3.7/αα  and in a homozygous state, -α3.7/-α3.7 .201 HbS heterozygotes, 36 HbC heterozygotes and 2 HbCS double heterozygote were detected from the 1,186 adults. The overall allele frequencies of HbS and HbC were 17.1%  and 3.1% , respectively. Similar to other sub-Saharan African regions, Bioko Island showed a high prevalence of αTo propose effective public health strategies, reliable epidemiological information for the local population must be obtained. The recent medical communication between Equatorial Guinea and China resulted in an opportunity to address important public health problems, including malaria control and erythrocyte genetic disorders on Bioko Island. This report represented the first detailed population survey assessing the frequencies of classical erythrocyte genetic disorders in this region.- variant five times longer than with the previously recommended 0.75 mg/kg dose [P. falciparum malaria in the region.Our survey revealed a high prevalence 8.64%, 359/4,144) of G6PD deficiency on Bioko Island. As compared with other African regions, the prevalence of G6PD deficiency was lower than that of Burkina Faso 31.0%) , Congo . The estimated number of pregnancies each year in which the fetus would be at risk for HbS homozygote (HbSS) was 69 . Alpha-talassemia 3.7-kb delection (hetero- and homozygote) was highly prevalent among these participants (52.4%). Indeed, because the Bioko Island Malaria Control Project, started in 2004, had a marked impact on malaria infection and mortality, and malaria was slowly decreasing on the island [Sickle cell disorders were frequent on the island, which was consistent with other findings , 37. Mode island ,39, the P. falciparum malaria in this region. An obligatory newborn screening program, prenatal screening and counseling for these genetic disorders, especially HbS, are needed on Bioko Island.In a conclusion, a high frequency of alleles such as G6PD deficiency, HbS, HbC and α-thalassemia associated with malaria resistance were present on Bioko Island. High frequency of G6PD deficiency indicated that the diagnosis and management of G6PD deficiency was necessary on Bioko Island. Before G6PD test was available, a 0.25 mg base/kg PQ single dose was recommended instead of 0.75 mg base/kg PQ dose to treatment regimens for


Preventing malaria during pregnancy is important for the health of mothers and newborns. Interventions, which include distribution of bed nets and administration of intermittent preventive treatment (IPT), typically occur at the first antenatal visit, usually in the second or third trimester of pregnancy. In 2012, during the course of ongoing clinical studies of malaria among pregnant women in Malawi, a universal bed net campaign was implemented by the Government. This study tested the hypothesis that a universal bed net campaign would decrease the prevalence of malaria among pregnant women at their first antenatal visit.Some 1661 women were recruited for two studies from 2009 to 2014. Quantitative PCR (qPCR) was conducted from dried blood spots collected at the first antenatal care visit (prior to administration of IPT or any study interventions) from women who were in their first or second pregnancy and less than 28 weeks gestation by clinical assessment.Overall, 320 of 1629 (19.6 %) women tested for malaria at their first antenatal visit were infected. Malaria infection rates declined from 28.4 % before the universal bed net campaign, to 18.5 % in 2012, to 15.0 % in the years following the universal bed net campaign. The odds of malaria infection at the time of first antenatal visit in 2012 and the years following the bed net campaign were significantly lower than in the years prior to the intervention . A similar pattern was observed for the prevalence of clinical malaria. The inverse trend was observed for reported bed net use. However bed net use and malaria infection were not significantly associated on the individual level.Malaria infection in pregnant women is common even after a bed net campaign in Malawi, though prevalence rates declined. These early infections may cause maternal anaemia and placental malaria resulting in adverse maternal and fetal outcomes. Infection early in pregnancy may also contribute to malaria transmission as pregnant women represent a significant untreated reservoir of parasites. Universal bed net distribution appears to have moderate success in preventing malaria early in pregnancy and these findings support continued efforts to target women early in pregnancy and all women of childbearing age. Every year 125 million pregnancies occur in malaria-endemic countries . To prevBed nets are typically distributed via antenatal and immunization clinics in order to reach high-risk populations: pregnant women and children under 5 years old. Because women often enroll in antenatal care late in pregnancy, interventions targeting all women of child-bearing age have the potential to protect against malaria early in pregnancy. In 2012, Malawi launched a universal bed net campaign. In Blantyre, the site of this study, the campaign targeted 250,000 households and distributed more than 400,000 bed nets with the goal of providing a bed net for every two individuals in a household. On a national level, in 2010 58 % of households had an ITN; in 2012 55 % of households had an ITN; and in 2014 70 % of households had an ITN. Universal ITN coverage  expanded from 19 to 30 % between 2012 and 2014 , 19. ExpTo test the hypothesis that a universal bed net campaign would reduce the burden of pregnancy-associated malaria prior to antenatal clinic attendance, data on bed net use and blood samples were collected at the first antenatal visit in clinical studies that occurred from 2009 to 2014 in Blantyre, Malawi. Malaria prevalence and reported bed net use were compared over this time period.Data presented are from the first antenatal visit from a sequential series of studies of pregnant women conducted in Ndirande, a peri-urban township of Blantyre, Malawi. Malaria transmission occurs year-round with a seasonal peak during the 3- to 4-month rainy season. In both studies, women were screened for enrolment at their first antenatal visit if they were in their first or second pregnancy, had not received IPT and were at less than or equal to 28 weeks of gestation. Women were excluded if they had any major illness requiring high-risk obstetric care, HIV infection or chronic antibiotic treatment. The first study was an observational study of malaria in pregnancy that enrolled women from June 2009 to June 2010 . The secPlasmodium falciparum lactate dehydrogenase or 18 s rRNA to detect infection. Extraction and PCR protocols are described online [Medical history and bed net use were recorded and physical examination, including axillary temperature, was performed. For all subjects, regardless of symptoms, finger-prick filter papers were collected and subjected to quantitative real-time PCR (qPCR) for d online . If the Ethical approval was obtained from the University of Malawi College of Medicine’s Research Ethics Committee and the University of Maryland Baltimore Institutional Review Board. Written informed consent was obtained from all participants before conducting any study-related activities. Participants had the option to withdraw from the study at any time. All data were recorded and analysed anonymously.The rainy season was defined as 1 December to 31 March and the dry season was defined as 1 April to 30 November. Clinical malaria was defined as parasites detected by light microscopy and associated symptoms including at least one of the following: objective fever measured at the clinic, history of fever in the previous 48 h or other symptoms in the previous 48 h including headache, myalgia, vomiting, or weakness. Malaria infection was defined as parasites detected by molecular methods, regardless of symptoms. Bed net use was determined based on the participant’s report of having slept under a bed net the previous night. Women also reported the number of nights in the previous month that they slept under a bed net. Frequency of bed net use was classified into three categories: slept under a bed net every night in the last month, slept under a bed net some nights in the last month, or never slept under a bed net during the last month. Gestational age was estimated based on last menstrual period for the 2009–2010 study and based on ultrasound for the 2012–2014 study.Data analysis was performed using Stata version 12.0 software . Graphs were produced using GraphPad Prism version 5.01 for Windows . Descriptive statistics were analysed using frequencies and proportions for categorical data and geometric means, means, standard deviations, and 95 % confidence intervals for continuous variables; 95 % confidence intervals were calculated for all proportions and prevalence rates. To measure the association between gravidity, maternal age, gestational age, seasonality or bed net use and malaria infection or clinical malaria, logistic regression was used to calculate odds ratios. P > 0.05 was considered statistically significant.Some 1661 women were screened at their first antenatal visit for two studies from 2009 to 2014. Data on bed net use were available for 1347 individuals. Overall, 57.8 % (779/1347) of women reported using a bed net at their first antenatal visit. Bed net use increased from 50.3 % (225/447) before the universal campaign, to 54.8 % (200/365) the year of the campaign, to 66.2 % (354/535) following the campaign . Malaria prevalence over time followed the inverse pattern of bed net use. Malaria prevalence declined significantly from 28.4 % (125/440) prior to the universal bed net campaign, to 18.5 % (87/471) the year of the campaign, to 15.0 % (108/718) following the campaign  of women had clinical malaria at their first antenatal visit. The prevalence of clinical malaria dropped significantly from 8.7 % (39/449) before the bed net campaign, to 3.0 % (11/364) the year of the bed net campaign and remained at 3.0 % (16/534) following  when analyzing individuals. There was no significant association between malaria infection and bed net use . There was also no significant association between gravidity, gestational age or maternal age and malaria infection or clinical malaria in univariate analysis (Table These data show significantly increased reported bed net use, decreased malaria infection, and decreased clinical disease prevalence at first antenatal visit following a universal bed net campaign. However, despite the universal bed net campaign, malaria prevalence at first antenatal visit remained high. Overall, one in five women was infected at the time of first antenatal visit, and even in the rainy season of 2013–2014, when infection prevalence was lowest, 13 % of women tested positive even in this urban setting. A clear inverse relationship was observed between bed net use and malaria infection. The lowest infection prevalence and highest bed net use were reported in the same year. However, bed nets did not appear to provide individual level protection against malaria. This suggests that the benefit of bed net use occurs on the population level as has been noted previously in settings where community bed net use is high .There are several limitations to this study. It relied on self-reported bed net use, which may have been inaccurate. While this may result in an overestimate of bed net use, this potential bias would be consistent across years and not affect the pre- and post-intervention patterns observed. All of the women enrolled were in their first or second pregnancy, and therefore at higher risk of pregnancy-associated malaria than multigravid women. Thus, the malaria infection prevalence may not be generalizable to all pregnant women. Conversely, because women presenting to the antenatal clinic after 28 weeks gestation and women not seeking antenatal care—groups with the most limited health care access—were excluded, the study population may be biased towards women with the most access to health care and likely to have lower rates of infection.The observation of the association between bed net use and decreased malaria prevalence does not prove causality. It is difficult to examine ecological data where interventions are applied across an entire country. However, given the documented benefits of bed net use during pregnancy –25, obseAlthough observational in nature and subject to the limitations described above, these results suggest a strong link between bed net use and prevalence of malaria in the pregnant population. There was a consistent inverse relationship between bed net use and malaria infection prevalence, including in 2014 when bed net use declined and malaria infection prevalence increased. This is consistent with intervention fatigue following a one-time campaign and the limited lifespan of bed nets. These data are mirrored by nationwide data on malaria prevalence in children under five collected as part of the Malaria Indicators survey in which microscopy-detectable malaria fell sharply from 43 % in 2010 to 28 % in 2012 and then rose slightly to 33 % in 2014 .Anopheles bites as a result of widespread availability of bed nets in the community, leading to lower transmission throughout the area.The fact that there is no individual-level association between malaria infection and bed net use is consistent with a population-level effect as has been demonstrated in other studies of bed net distribution , 28. PrePrevious studies have clearly demonstrated the need to reach pregnant women early in their pregnancy, before malaria infection occurs. This is challenging since it is typical for women in sub-Saharan Africa to delay antenatal care until late in pregnancy , 10 poteA universal bed net campaign was associated with a reduced prevalence of malaria during pregnancy at the first antenatal visit. These results suggest that the expansion of interventions implemented universally may be required to reduce the burden of pregnancy-associated malaria.


Plasmodium spp. infections were detected by PCR in 17.4%  of blood samples that microscopy had shown to contain only P. falciparum. The quality of microscopy must be improved because use of PCR for detection of malaria parasites is limited in rural areas.In 8 malaria-endemic states in India, mixed  Plasmodium species  cause human malaria. Malaria is not uniformly distributed in India; 8 of the 35 states and union territories contain most malaria cases  are useful detection tools . Written informed consent was obtained from all participants or parents of children, according to Indian Council of Medical Research guidelines.The study was conducted in 2 community health centers (CHCs), 1 in an area that had a high level of malaria endemicity and 1 that had a low level of malaria endemicity, in each of 8 states in India: Orissa, Chhattisgarh, Jharkhand, Maharashtra, Madhya Pradesh, Tripura, Gujarat, and Rajasthan ; Table 1Blood samples were collected from persons with suspected malaria during July–December 2014 at malaria clinics in CHC hospitals at 15 sites. Microscopy and RDTs  were performed at outpatient department clinics of CHCs, and molecular diagnosis (PCR and sequencing) was performed at the molecular parasitology laboratory at the National Institute for Research in Tribal Health.For microscopy, thick and thin blood smears were prepared from finger prick blood samples, which were air-dried, fixed in methanol, and stained with Giemsa. A total of 100 thick blood smear fields were examined by using an oil immersion lens at 100× magnification before a sample was considered negative. Malaria parasite density was determined from thick blood smears by counting the number of parasites against 200 leukocytes . Species-specific nested PCRs that targeted the 18S rRNA gene were used to detect 4 malaria parasite species   was performed as described . However, PCR showed mixed infections with P. falciparum and P. vivax in 239 (16%) samples; P. falciparum and P. malariae in 19 (1%) samples; P. falciparum and P. ovale in 6 (0.4%) samples; and P.falciparum, P. malariae, and P. ovale in 1 (0.1%) sample  sample . MicroscP. falciparum and P. vivax. PCR analysis showed that the highest prevalence of mixed infections with P. falciparum and P. vivax was in Jharkhand , followed by Madhya Pradesh , Rajasthan , Orissa , and Tripura , and Chhattisgarh . The lowest prevalences were in Maharashtra  and Gujarat .Secondary microscopic analysis of blood smears by a second technician showed that only 22/239 (9.2%) samples contained mixed infections with P. falciparum and P. malariae were found in all 8 states, although in small numbers. Mixed infections with P. falciparum and P. ovale were found in only 4 states, particularly at CHCs in areas to which malaria was highly endemic. Of 7 mixed infections that contained P. ovale, 5 contained P. ovalecurtisi and 2 contained P. ovalewallikeri. P. knowlesi was not found in any state.Mixed infections with P. falciparum) and 70% of deaths caused by malaria in the entire country .This study was conducted 8 states in India that contain 80% of malaria cases (85% of which are caused by Our study had some limitations. Monoinfections or mixed infections were not verified by PCR if parasitemia levels were too low to be detected by microscopy or RDTs. Thus, mixed infections with parasitemia levels below the limit of detection of microscopy or RDTs would not have been detected. Detailed studies in different ecosystems during different transmission seasons and large sample sizes are required for a more accurate picture of mixed infections with common and uncommon parasite species, clinical epidemiology, adverse effects, relapse, and recrudescence.P. vivax, P.malariae, and P. ovale, which are not detected accurately by microscopy or RDTs. Although P.vivax and P. ovale are responsible for relapses (P. malariae is sustained at low rates among sparse and mobile human populations for decades, thus facilitating transmission by mosquitoes (Our results highlight the role of mixed infections, particularly those with


During pregnancy, women living in malaria-endemic regions are at increased risk of malaria infection and can harbour chronic placental infections. Intermittent preventive treatment with sulphadoxine-pyrimethamine (SP-IPTp) is administered to reduce malaria morbidity. It was hypothesized that the presence of placental malaria infection and SP-IPTp use would increase the risk of peripheral blood gametocytes, the parasite stage that is transmissible to mosquitoes. This would suggest that pregnant women may be important reservoirs of malaria transmission.Light microscopy was used to assess peripheral gametocytaemia in pregnant women enrolled in a longitudinal, observational study in Blantyre, Malawi to determine the association between placental malaria and maternal gametocytaemia. The relationship between SP-IPTp and gametocytaemia was also examined.2,719 samples from 448 women were analysed and 32 episodes of microscopic gametocytaemia were detected in 27 women. At the time of enrolment 22 of 446 women (4.9%) had gametocytaemia and of the 341 women for whom there was sufficient sampling to analyse infection over the entire course of pregnancy, 27 (7.9%) were gametocytaemic at least once. Gametocytaemia at enrolment was associated with placental malaria, defined as malaria pigment or parasites detected by histology or qPCR, respectively , but was not associated with adverse maternal or foetal outcomes. Administration of SP-IPTp did not affect gametocyte clearance or release into peripheral blood.Gametocytaemia is present in 5% of pregnant women at their first antenatal visit and associated with placental malaria. SP-IPTp does not alter the risk of gametocytaemia. These data suggest that pregnant women are a significant reservoir of gametocyte transmission and should not be overlooked in elimination efforts. Interventions targeting this population would benefit from reaching women prior to first antenatal visit. Malaria is a major cause of morbidity and mortality worldwide and it disproportionately affects children and pregnant women. Every year, 125 million pregnancies occur in malaria-endemic countries . Women bIn addition to placental sequestration increasing the risk of malaria infection and gametocytaemia, anti-malarial interventions may impact the transmission potential of pregnant women. The current strategy to protect against the adverse effects of placental malaria is intermittent preventive treatment in pregnancy with sulphadoxine-pyrimethamine (SP-IPTp) given two to three times during pregnancy . HoweverTo test the hypothesis that pregnant women serve as reservoirs of malaria transmission in malaria-endemic settings, the prevalence of gametocytaemia during pregnancy was measured in an observational study. The observational study reflected the natural history of gametocytaemia in pregnant women in a malaria-endemic area who received standard dosing of SP-IPTp. The association between placental infection or SP-IPTp and the presence of gametocytes was assessed.Pregnant women were offered enrolment in an observational study of pregnant women conducted in Ndirande, a peri-urban township of Blantyre, Malawi. Malaria transmission occurs year-round with a seasonal peak during the three- to four-month rainy season. Women were enrolled at their first antenatal visit if they were in their first or second pregnancy and at less than or equal to 28 weeks of gestation. Women were excluded if they had any major illness requiring high-risk obstetric care, HIV infection or chronic antibiotic treatment. Women were followed through delivery. Details of the study design and primary study results have been published previously . GestatiWomen attended routine clinic visits every four weeks and were encouraged to return any time they were ill. At all visits, including routine visits and whenever there was an illness with symptoms suggestive of malaria, history and physical examination were performed, with axillary temperature recorded and a fever defined as ≥37.5°C. Thick and thin malaria smears were collected and drops of blood were collected on filter paper. Malaria smears were field-stained and examined using light microscopy with a 100× oil immersion lens. Parasitaemia was assessed by counting the number of parasites per 200 leukocytes and examining 100 high-power fields before considering a smear to be malaria-negative. All slides were read by two microscopists and in cases of disagreement between the readings, the conflict was adjudicated by a third expert reader. Parasite density was calculated based on 8,000 leukocytes per microlitre. All smears identified as having parasitaemia in the observational study were classified as having asexual-stage infection and were treated with artemether-lumefantrine.Participants were given SP-IPTp up to three times in pregnancy during prenatal follow-up, in accordance with national policy in Malawi. At delivery the placenta was collected for molecular and histopathological examination. Newborns were assessed and gestational age was estimated as previously described . HaemoglPlasmodium falciparum lactate dehydrogenase to detect infection. Extraction and PCR protocols are described online [After completion of the observational study, dried blood spots collected on filter paper from all antenatal visits were subjected to quantitative real-time PCR (qPCR) for d online . A peripPlacental tissue biopsies were processed as previously described  and examDried blood spots and corresponding blood smears were examined from all women at enrolment prior to the administration of SP-IPTp. To determine the prevalence of gametocytaemia during pregnancy, data were included from all women who had gametocytaemia at any time during pregnancy. Women were classified as not having gametocytaemia during pregnancy if they had specimens available for analysis from enrolment and at least two months of follow-up. Data from one placenta of twin gestations were included in analyses of placental malaria but excluded from analyses of birth outcomes. When placental results were discordant, only the positive placental result was included.To determine the effect of SP on gametocyte clearance, all women who were considered malaria-negative in the observational study (and therefore not treated with artemether-lumefantrine) but subsequently found to be gametocyte-positive at enrolment were included. To examine the effect of SP on gametocyte release, women who were gametocyte-negative by microscopy but had sub-microscopic infection  at the time of enrolment were evaluated. In both analyses, the proportion of women with gametocytaemia at their visit one month after enrolment was compared using Fisher’s exact test.Data analysis was performed using STATA version 12.0 software . Descriptive statistics were analysed using frequencies and percentages for categorical data and geometric means, means, medians, and 95% confidence intervals for continuous variables. 95% confidence intervals were calculated for all proportions and prevalence rates. To measure prenatal risk factors and outcomes associated with gametocytaemia, logistic regression was used to calculate odds ratios. For the multivariate model, any parameter in the univariate model associated with gametocytaemia with p ≤ 0.1 was considered. Maternal treatment for malaria prior to enrolment, asexual stage parasitaemia at enrolment, mean maternal haemoglobin at enrolment, haemozoin in the placenta, and parasites detected in the placenta by qPCR were included. All p-values are two-sided and statistical significance was set at p ≤ 0.05.A total of 2,719 blood smears from 448 women were available and read for gametocytes if the corresponding filter-paper blood spot was qPCR-positive for malaria. Thirty-two episodes of gametocytaemia in 27 women were detected. Twenty-two (68.7%) of the gametocyte episodes occurred at the enrolment visit and all episodes of gametocytaemia occurred within two months of enrolment. Five women had gametocytaemia twice during their participation in the study. The geometric mean gametocyte density among gametocyte-positive smears was 19.9 gametocytes per microlitre of blood (95% CI: 13.3-29.8).vs 10/386, 2.6%). Among the 341 participants who had sufficient data to evaluate the presence of any gametocytes during pregnancy, 27 (7.9%) had at least one episode of gametocytaemia. The proportion of women with gametocytaemia during pregnancy was higher among women who had asexual-stage malaria detected by light microscopy at least once during pregnancy, 18 of 65 (27.7%), as compared to nine of 276  for those without asexual-stage infection detectable by microscopy.At enrolment, 22 of 446 (4.9%) smears were positive for gametocytes. Enrolment smears from two women were damaged and could not be evaluated. The gametocyte prevalence was approximately ten-fold higher among those women with asexual-stage malaria detected by light microscopy compared to those without infection detected by light microscopy  women had evidence of placental malaria either by histology, qPCR or both. Among the 311 women with both histology and qPCR data available, 33 (10.6%) had placental parasites alone, 38 (12.2%) had haemozoin alone, 33 (10.6%) had both parasites and haemozoin, and 207 women had neither. Of the ten placentas for which histology was unavailable, six (60%) had placental malaria by qPCR.At enrolment, gametocytaemia was associated with placental malaria, lower maternal haemoglobin and the presence of asexual stage infection. Age, gravidity, gestational age, history of malaria treatment, maternal temperature at enrolment and bed net use were not associated with gametocyte infection Table . No womeGametocytaemia at enrolment or at any time during pregnancy was not associated with differences in gestational age at birth, birth weight or maternal haemoglobin concentration at delivery.vs 6.1%, p = 0.5, Figure When examining women who had gametocytaemia at enrolment, all women cleared gametocytes within one month regardless of whether they received SP Figure A. Among These data show 5% gametocyte prevalence at the time of first antenatal visit and increased odds of peripheral gametocytaemia among women with evidence of placental malaria at delivery. This association was independent of asexual stage parasitaemia. When examined separately in a multivariate model, the presence of haemozoin, was associated with gametocytaemia at the time of enrolment and the association with active placental malaria infection did not achieve statistical significance (p = 0.09). In the context of SP-IPTp and active case detection and treatment with artemether-lumefantrine, episodes of gametocytaemia were rare after initiating antenatal care. All episodes of gametocytaemia occurred within two months of enrolment. No association between SP and either gametocyte release into peripheral blood or gametocyte clearance was found.This is the first study examining the relationship between placental malaria and gametocytaemia, and one of the few studies examining gametocytaemia in the context of pregnancy. A previous study in Tanzania documented a high prevalence of gametocytaemia (12-23%) in pregnant women with microscopically confirmed malaria . This isThe prevalence of gametocytaemia among pregnant women in this study is similar to that of children in the region, suggesting that pregnant women serve as a potentially important reservoir of malaria transmission, just as malaria-naïve children do. The prevalence of gametocytes among children with symptomatic uncomplicated malaria was 22.6% in Tanzania , which iA direct comparison cannot be made between pregnant and non-pregnant adults due to a lack of data on non-pregnant adult gametocyte prevalence in Malawi and neighbouring countries. However, it has been observed in multiple African settings, that with increasing age, gametocyte prevalence drops rapidly , 24.Although the rates of gametocytaemia are similar between children and pregnant women, the transmission potential of pregnant women raises unique concerns. Their infections are almost always asymptomatic—as was the case for all women in this study at the time of enrolment—whereas children are more likely to exhibit symptoms of malaria leading to treatment intervention, potentially reducing their transmission potential. Although young children contribute a larger proportion of the transmission reservoir than pregnant women , the ideet al.[There are several limitations to this characterization of gametocyte prevalence. All of the women enrolled were in their first or second pregnancy, and therefore at higher risk of pregnancy-associated malaria and placental malaria than multigravid women. Thus the gametocyte prevalence in the general pregnant population may be overestimated. However, because this study was limited to detecting gametocytes by light microscopy, the results likely underestimate the true prevalence of gametocytes. Light microscopy is insufficiently sensitive to detect all infections that are transmissible to mosquitoes. Comparing molecular methods with microscopy, Bousema et al. observedet al.. In addiin vitro to increase gametocytogenesis [The high rate of gametocytaemia and its association with placental malaria infection suggests that pregnancy, and possibly the presence of placental sequestration put pregnant women at risk for gametocytaemia. Gametocytes are very rarely found in the placenta , making ogenesis . The incIn this limited sample size, no association between SP prophylaxis and gametocytaemia was detected. This is consistent with a previous study demonstrating that SP-IPT in children decreased gametocytaemia . The incMost cases of gametocytaemia in this study occurred at the time of enrolment, suggesting that pregnant women may be an important source of malaria transmission early in pregnancy. This has significant public health implications. It highlights the need for interventions aimed at preventing malaria to reach women earlier in pregnancy. This study and others , 19 haveIt is also possible that maternal gametocytaemia could impact foetal and infant immunity. Gametocytaemia could result in increased production of gametocyte-specific antibodies that would be transferred to the infant providing protection against gametocyte carriage in infancy. Like asexual-stage proteins , gametocThe results of this study raise the possibility that activity against gametocytes may be important in the selection of strategies to prevent and control pregnancy-associated malaria. Although all cases of gametocytaemia were cleared within three months of participation in the study, study interventions were extensive, including SP-IPTp, which is not gametocidal, and treatment with artemether-lumefantrine, which is known to have some activity against gametocytes. This study was not able to evaluate the relationship between artemether-lumefantrine treatment and gametocytaemia. It is possible that clearance of asexual stages via IPTp is sufficient to prevent gametocyte development from the asexual stages, and thus prevent gametocytaemia. Alternatively, the treatment targeting asexual stages may allow gametocytes to remain in the periphery and bone marrow, or may even promote gametocytogenesis. IPTp with drugs with known gametocidal activity, such as the artemisinin derivatives may be necessary to prevent parasite transmission. Further study is needed to elucidate the mechanism of gametocytogenesis and determine which drugs are the most effective in clearing infections and also preventing transmission. The results of this study highlight the critical role of preventing malaria in pregnant women, not only to protect the health of the mother and the infant but also to make progress towards malaria elimination.Gametocytaemia was observed in 5% of pregnant women before they initiated antenatal care and began receiving IPTp. Placental malaria is associated with increased odds of gametocytaemia in pregnancy independently of asexual stage parasitaemia. In this study, there is no evidence that SP-IPTp increases the presence of gametocytes. Overall these data suggest that pregnant women may be a significant reservoir of gametocytes and should not be overlooked in malaria elimination strategies.