Malaria is still a public health problem in Sub-Saharan Africa. Malaria causes mortality mostly in children under-five years. Early detection and prompt treatment using recommended antimalarials is key to malaria control. However, in Tanzania, contrary to the national goals, a large proportion of children with fever taken to health facilities are not tested for malaria and those tested positive are not promptly treated using recommended antimalarials. The aim of this study was to determine factors associated with malaria testing and prompt use of recommended antimalarials among under-five children with fever in Tanzania.This was a secondary analysis of Tanzania HIV and Malaria Indicator Survey (THMIS) data 2011-12 obtained from a national cross sectional survey. The analysis involved children aged 6-59 months whose mothers reported they had fever two weeks preceding the survey. Factors associated with testing and uses of recommended antimalarials were obtained using logistic regression.Of the 1675 under-five children with fever, 951 (56.8%) were taken to the health facilities. Of the 951 children, only 394 (41.48%) febrile children were tested for malaria. Of those tested, 291 (78.91%) were diagnosed with malaria. Of those diagnosed with malaria, only 124 (42.68%) children used recommended antimalarials within 1st 24 hours of diagnosis. In multivariate analysis, children taken to health centers  and to the hospitals  had higher odds of being tested compared to those taken to dispensary and other lower level health facilities. Children were more likely to use recommended antimalarial promptly if they had a caretaker with secondary or higher education  or living in the rural area  compared to those with an uneducated caretaker or from an urban area.Training on malaria testing and treatment guidelines should be provided, and preventing stock outs of malaria testing kits and medications at dispensary level should be made available as it is the first point of health care for most Tanzanians. Reasons on why urban people are less likely to use recommended antimalarials need to be investigated and addressed for proper malaria management. Malaria is still a public health problem especially in SSA . It killIn Tanzania, approximately 90% of the population lives within five kilometers of a health facility . HoweverDespite the introduction of mRDT in malaria diagnosis in 2011, in Tanzania, contrary to the national goals  and thosWhile studies have assessed factors influencing testing for malaria among children; facility factors , cliniciThis study analyzed the Tanzania HIV and Malaria Indicator Survey (THMIS) of 2011–12, which is the most recent in a series of country surveys, with the following objectives; 1) to determine factors associated with malaria testing and 2) to determine factors associated with prompt use of recommended antimalarials following malaria diagnosis among under-five year children taken to health facilities by their caretakers.This is a secondary analysis of data from Tanzania HIV and Malaria indicator survey (THMIS) 2011–2012 which is the third and most recent in a series of national sample surveys that aims to estimate the key indicators of malaria and HIV for each Tanzania’s region.THMIS is a nationally-representative cross-sectional household surveys that are performed in an interval of four years. The information can be generalized from the region, zonal and national level, and not below these levels. Sample was obtained by using two stage probability sampling, first stage involved selection of clusters and second stage selection of households. Clusters were selected from a list of enumeration areas of the 2002 Population and Housing Census. In the 2011–12 THMIS 583 clusters were selected from which 10,496 households participated in the survey. In total, 8648 mothers with under-five children were interviewed.In individual questionnaire, female respondents were asked questions to collect information on several topics including but not limited to; socio-demographic characteristics, sexual and reproductive history, and on knowledge of malaria and HIV. Information about recent fever and treatment of fever for children who were less than five years old was also collected during interviews as well as place of care and treatment received. The Household Questionnaire also collected information on characteristics of the household to determine the wealth index of households, ownership of mosquito nets and recording biomarkers test results, anemia for children under-five years old .The data set which was used is that with children’s information i.e. Children Records (KR dataset). The unit of analysis in this study was children under five (5) years old (age 6–59 months) and their mothers.Testing for malaria variable was obtained based on the reported information from the caretaker that children with fever two weeks preceding the survey were taken to the health facility, had blood taken either from a heel or a finger. Prompt use of recommended antimalarials- this variable was created by labeling those who used recommended antimalarials within 24 hours of diagnosis, as shown in the Tanzanian Malaria Diagnosis and Treatment guidelines. Children who were positive for malaria but used antimalarial after twenty four hours were labeled otherwise. Wealth index- this is a re-coding of the wealth index variable present in the dataset in three categories contrary to the five categories present in the dataset. This variable was created by compressing the original wealth index into tertile. Instead of poorest, poor, middle, rich, richest the newly re-coded wealth index was put into poor, middle and rich categories.STATA Version 12  was used in data processing and analysis accounting for the complex sampling design of the THMIS, clustering effect. Descriptive analysis included computations of frequency and percentages for categorical variables and measures of central tendency and respective measures of dispersion for continuous variables. Using Logistic Regression, crude and adjusted Odds Ratios were obtained to assess the strength of association between independent variables and the outcome variables. P-value of less than (5%) with 95% Confidence interval were used to measure statistical significance of the association between independent and dependent variables.Denominator for analyzing factors associated with testing for malaria were determined by analyzing children who were reported to have fever two weeks preceding the survey and whose mothers sought care to health facility. The denominator for prompt use of recommended antimalarials were obtained by analyzing children who were reported to have fever two weeks preceding the survey, whose mothers sought care to health facility and tested positive for malaria.During data collection, the broad goals of the exercise were explained to the respondents by fieldworkers during their introduction in the household and written consent was obtained from all participants prior to all the data collection procedures . The ethOf the 8573 children studied, 1675 had fever two weeks preceding the survey. Of these 1675, 51% were males. The median age of children included in this study was 23  months. The majority of the 1675 children were residing in rural 1359 (81.1%) and from families with low wealth index 790 (47.2%), Of the 1675 febrile children, 1312 (78.3%) were given care or taken for health care at pharmacies, health facility or treated at the household level. In this analysis health facilities are taken to be appropriate place for care seeking of children with fever because malaria suspected cases can be checked by mRDT and can be appropriately managed. Nine hundred and fifty one children (56.76%) were taken to appropriate health facilities , had appropriate healthcare seeking behavior (AHCSB). Of the 951 febrile children whose mothers sought appropriate healthcare, 394 (41.48%) were tested for malaria. Of the 394 febrile children tested, 291 (78.91%) tested positive for malaria. Of the 291 malaria positive children, 266 (91.35%) children were treated using any antimalarials. Two hundred and nine (71.72%) children were treated using recommended antimalarials (which was artemether-lumafantrine ALu) any time after being diagnosed. Of the 291 children diagnosed with malaria, only 124 (42.68%) children used recommended antimalarials promptly i.e. the same or next day after being diagnosed with malaria as shown in Of the 124 children who used recommended antimalarials, 74 (59.68%) used Artememther Lumefantrine, followed by Quinine 44 (35.48%), three children (2.42%) diagnosed with malaria used Artesunate and Amodiaquine and 3 (2.42%) used Artesunate. Substantial number of malaria cases 33 (11.34%) got treated using regimen not recommended on the guideline by 2011 . Others, 5 (1.72%) used antimalarials which were not specified while 25 (8.59%) others used nothing after being diagnosed with malaria.Of the 556 (58.46%) children with fever who were taken to appropriate healthcare facilities but not tested, 305 (32.07%) got treated using any antimalarials anytime while of these, 167 (17.56%) used recommended antimalarials promptly.Children living in rural areas have 79% less odds of being tested compared to those in urban areas . Also, children taken to health centers and hospitals have high odds of being tested compared to those taken to dispensary and other lower levels. The odds of testing are 1.8  and 3.4  times higher for children taken to health centers or hospitals respectively compared to those taken to dispensary and other lower level health facilities, Urban residence, having not-working caretaker and caretaker with secondary or higher education have been shown to be associated with prompt use of recommended antimalarials following malaria diagnosis (P<0.05).Children living in rural have three times higher odds of using recommended antimalarials promptly following malaria diagnosis compared to their urban counterparts  while, children with caretaker who have secondary education and more have four times higher odds of using recommended antimalarials promptly following malaria diagnosis compared to those with caretaker with no education . For children with not-working caretaker, the odds of using recommended antimalarials promptly following a diagnosis of malaria is four times higher compared to those children with working caretaker , This study determined factors associated with testing and prompt use of recommended antimalarials. The results show that still there is substantial number of febrile children under-five years that are not tested for malaria despite being taken to the health facilities and very few of those diagnosed with malaria use recommended antimalarials and within recommended time.Furthermore, the results showed factors associated with malaria testing are residence and type of the health facility febrile children were taken to while factors associated with prompt use of recommended antimalarials after being diagnosed with malaria are residence, working status of the caretaker and the education level of the caretaker.Less than 50% of the children with fever taken to appropriate health facilities were tested for malaria. This proportion is very low compared to the Tanzanian guideline for managing malaria which requires testing to be universal for every child presenting with fever at the health facilities . The lowOne of the goal in Tanzania medium term malaria strategic plan is to have at least 80% of positive malaria cases receiving recommended antimalarials within 24 hours after being diagnosed with malaria . But froFurther, more than 30% of the febrile children who were not tested for malaria used antimalarials. This finding is similar with the results reported in other parts of sub-Saharan Africa ,24,30. TIn this study, testing for malaria was done more at hospitals and health centers than at dispensaries. In Tanzania nearly 70% of the dispensaries are in rural areas where 80% of Tanzanians live, while most hospitals are in urban . This meThe strength of this study lies on the fact that the data is representative of the Tanzania population which strengthens the generalisability of the results. Also mothers were asked on a history of fever and its management within two weeks preceding the survey which decreases recalling bias. The weakness point of this study is that it analyzed only individual variables collected by THMIS survey but failed to explore other factors which plausibly are associated with outcomes of interest, malaria testing and prompt use of recommended antimalarials, for example health facility factors. The second limitation to this study is it cross sectional nature which is always good to develop hypothesis but cannot tell the causal relationship between independent factors and outcomes.To effectively control malaria, low proportion of malaria testing and low proportion of prompt use of recommended antimalarials following malaria diagnosis should be addressed through frequent training on guidelines, provision of malaria testing devices and close monitoring. Further studies are needed on why children in urban areas are not given recommended antimalarial despite being more frequently tested for malaria.


As malaria prevalence declines in many parts of the world due to widescale control efforts and as drug-resistant parasites begin to emerge, a quantitative understanding of human movement is becoming increasingly relevant to malaria control. However, despite its importance, significant knowledge gaps remain regarding human movement, particularly in sub-Saharan Africa.A quantitative survey of human movement patterns was conducted in four countries in sub-Saharan Africa: Mali, Burkina Faso, Zambia, and Tanzania, with three to five survey locations chosen in each country. Questions were included on demographic and trip details, malaria risk behaviour, children accompanying travellers, and mobile phone usage to enable phone signal data to be better correlated with movement. A total of 4352 individuals were interviewed and 6411 trips recorded.cf 21 days, p < 0.001) compared to other travellers.A cluster analysis of trips highlighted two distinct traveller groups of relevance to malaria transmission: women travelling with children  and youth workers . Women travelling with children were more likely to travel to areas of relatively high malaria prevalence in Mali , Burkina Faso , Zambia , and Tanzania  compared to other travellers. They were also more likely to own bed nets in Burkina Faso  and Zambia , and less likely to own a mobile phone in Mali , Burkina Faso , and Zambia . Malian youth workers were more likely to travel to areas of relatively high malaria prevalence  and for longer durations  contains supplementary material, which is available to authorized users. As a vector-borne disease, malaria is spread alternately between its human and mosquito hosts. Humans travel much larger distances than mosquitoes and consequently human movement plays a dominant role in parasite dispersal , 2. SignDespite the importance of human movement data in planning disease control, significant data gaps remain , particuSubstantial qualitative work has been conducted describing the types of human movements in Africa, many of which are relevant to malaria transmission. For example, in the Sahel, a semi-arid region beneath the Sahara Desert including parts of Mali and Burkina Faso, seasonal rains are correlated with both agricultural labour movements and malaria transmission due to an abundance of vector-breeding sites , 15. RurAnopheles gambiae and Anopheles funestus, bite at night. Questions were also asked about demography and trip details to gain a better descriptive understanding of movement, about mobile phone usage to enable phone signal data to be better correlated with self-reported movement patterns, and about malaria risk behaviour and children accompanying travellers, since children are the most common parasite carriers.To gain a better quantitative understanding of these patterns, a human movement survey was conducted in four countries with ongoing malaria transmission: two in West Africa , one in East Africa (Tanzania) and one in Southern Africa (Zambia) Fig. . In consThe survey was undertaken in four sub-Saharan African countries with ongoing malaria transmission—Mali, Burkina Faso, Zambia, and Tanzania Fig. . In MaliThe survey sites were chosen in collaboration with local malaria researchers and medical anthropologists. These were sites that: (a) were judged to capture a wide range of traveller groups present in the country; and, (b) had an existing relationship with the local researchers. Sampling of households within each study site was randomized, however, the study sites themselves were a judgement/convenience sample. Hence, inferences based on these results reflect the collection of study sites rather than the countries at large.The Mali survey was conducted in Bamako, the capital city and largest urban centre, two fishing villages in Baya, two farming villages in Baroueli, and in Mopti and Fatoma, a commercial centre and village, respectively, approximately 460 km northeast of Bamako. The Burkina Faso survey was conducted in Ouagadougou, the capital and largest city, and two nearby locations—Sapone, an agricultural village approximately 50 km south of Ouagadougou, and Boussé, a major centre of agriculture and trade approximately 50 km north of Ouagadougou. The Zambia survey was conducted in five locations—Lusaka, the capital and largest city; Samfya, a central fishing town; Kitwe, an urban trading town in the Copperbelt; Nakonde, a town in the northeast bordering Tanzania; and, Chipata, a rural town in the east bordering Malawi. The Tanzania survey was conducted in four locations—Dar es Salaam, the capital and largest city; Ifakara, a small rural town on the edge of the Kilombero valley; Muheza, a small rural town near the Indian Ocean and the border with Kenya; and, Mtwara, an agricultural town with a growing mining industry near the Indian Ocean and Mozambique.Participants were recruited within each location using random sampling techniques , basic demographic information , mobile phone usage , and malaria . Questions were also asked about migratory patterns. Details are provided in Additional file A trip was considered short-term if its duration was 2 weeks or less, and long-term if its duration was longer than 2 weeks but less than a year. Details were recorded for the three most recent short- and long-term trips. Travel within the ward, commune or city of origin was not included. Destinations were recorded at the level of commune or ward via dropdown lists in Epicollect. Dropdown lists were populated using shape files for each of the study countries, allowing locations to be geocoded. Study participants were interviewed in Mali during the rainy season of September/October 2010 and the dry season of March 2011, in Burkina Faso during the rainy season of July 2011, in Zambia during the cool dry season of July/August 2012, and in Tanzania during the long rainy season of March 2013.Centre National de Recherche et de Formation sur le Paludisme in Burkina Faso, ERES Converge in Zambia and the Ifakara Health Institute Research Ethics Board in Tanzania. The survey was anonymous and informed consent was obtained from all participants.Ethical approval for the study was granted by the Imperial College Research Ethics Committee, UK and the Institutional Review Boards of the Malaria Research and Training Center in Mali, the Q > 0.5 [A descriptive analysis of the demographic data, trip properties, mobile phone usage, behaviour, and malaria-related variables was undertaken using individual trips as the unit of analysis. Hierarchical cluster analysis was performed using trip properties  and the demographics of the travellers  as input variables in the FactoMineR package in R . All recQ > 0.5 .Multivariable logistic regression was used to identify differences in mobile phone usage behaviour and malaria-related variables between the identified trip clusters. Malaria-related variables included bed net ownership, bed net usage at the origin and destination of travel, perceived malaria risk, and estimated malaria prevalence at the origin and destination of travel. Perceived malaria risk was derived from the following survey questions—“Do you think you are at risk of getting malaria where you live?” and “Do you perceive there to be a risk of malaria where you travelled to?” with high, medium and low response options. Estimated malaria prevalence was based on spatially stratified 2010 estimates of parasite prevalence in 2–10 years old  aggregatcf 730 females, p = 0.002), while the Zambia sample had significantly more female interviewees . Interviewees in the Tanzanian sample reported more children under the age of five residing in the same residence than in the other countries  and interviewees in the Zambia sample reported more children under the age of five than in the Mali and Burkina Faso samples . Interviewees in the Burkina Faso and Zambia samples reported significantly more trips than interviewees in the Mali and Tanzania samples , while DHS surveys reported a higher proportion of the populations of Zambia and Tanzania as having travelled in the last year compared to the populations of Mali and Burkina Faso [cf 86 % for Tanzania/Burkina Faso, p < 0.001). Mobile phone ownership was significantly lower among interviewees in the Malian sample compared to the Burkinabe and Tanzanian sample . Differences between the rainy and dry season samples in Mali are described in Section 2.1 of Additional file In total, 4352 individuals were interviewed—1588 from Mali, 721 from Burkina Faso, 1093 from Zambia and 950 from Tanzania . For example, in the Mali sample, the mean trip distance for trips in the ‘women with children’ cluster was 120 km, compared to 157 km for those in other clusters, and in the Zambia sample, the mean trip distance was 161 km for the ‘women with children’ cluster, compared to 203 km for other trip clusters . For example, in the Burkina Faso sample, travellers in the ‘women with children’ cluster made a mean of 2.5 trips in the last year, compared to travellers in other clusters who made a mean of 5.7 trips  and involved larger distances . The vast majority of travellers in this group (87 %) had no children under the age of five.In the Mali sample, the cluster of trips that captured the second highest amount of variance in the data consisted predominantly of trips made by Malian youths between the ages of 16 and 29 years travelling predominantly for work-related reasons  and to have more children under 5 years . Travellers making trips in the ‘general’ cluster tended to make more overnight trips per year than those making trips in the ‘women with children’ and ‘youth worker’ clusters . Trips in the ‘general’ cluster also tended to cover larger distances than those in the ‘women with children’ cluster .The remaining trips belonged to a ‘general’ cluster. Travellers making trips in this ‘general’ cluster were predominantly male, belonged to all age groups, and trips in this cluster were for both family and work-related reasons Fig. . In the Trips in the ‘women with children’ cluster were significantly more likely to be to areas of high malaria transmission than those in the ‘general’ cluster in the samples from Mali , Burkina Faso , Zambia , and Tanzania  , Burkina Faso  and Zambia   and the Zambia sample . In the Burkina Faso sample, international trips were more frequently made by travellers belonging to the 30–45 years old age group . International trips were more likely to be for work-related reasons than national trips in all country samples  and, in the Burkina Faso sample, although international trips were less likely to be made by travellers owning a bed net , international trips were more likely to be made by travellers using a bed net on their trip . International trips were more likely to be made by travellers owing a mobile phone than national trips in all country samples . There were no significant differences in terms of children accompanying travellers for international or national trips. International trips tended to involve longer durations than national trips in the Mali sample  and the Burkina Faso sample , however, they were relatively shorter in the Zambia sample , possibly because several of the Zambian survey sites were border towns.The survey also captured cross-border movements that were not included in the within-country analysis Table . Cross-bFrom the Mali survey sites, international travel was most commonly reported to other West African countries, such as Côte d’Ivoire, Guinea, Senegal, Burkina Faso, and Ghana, with occasional trips to France, the former colonial power, and Saudi Arabia for religious worship . This could introduce a bias towards trips in the months preceding the interviews in two ways—recall bias, since recent trips may be easier to remember; and ‘trip clipping’ since, for people who have taken many trips, only the recent ones will be recorded. Furthermore, certain questions are subject to social desirability bias, since respondents may feel inclined to give the ‘right’ answer  .As a growing number of diseases are targeted for elimination, including poliomyelitis, Chagas disease and neglected tropical diseases, such as schistosomiasis, lymphatic filariasis and onchocerciasis, the role of travellers in sustaining transmission will become increasingly important. The combined survey and cluster analysis approach outlined here provides a powerful framework for identifying key traveller groups that, in combination with knowledge of the local epidemiology of disease transmission, will help to inform disease elimination programmes. Information on key traveller groups will also help to inform control programmes for emerging pathogens, such as Ebola , Zika 3, and patA study following this approach randomized at a national scale is needed to infer nationally representative traveller groups, however, the survey results analysed here provide a snapshot of traveller groups present in a collection of sites in Mali, Burkina Faso, Zambia, and Tanzania. These provide preliminary evidence for the generality of the ‘women with children’ traveller group across all four countries, despite significant differences in culture and geography. Results from the Mali survey also provide preliminary evidence for youth workers as a key traveller group, in agreement with previous qualitative studies , 15–17.Further survey work is encouraged, and questions recommended for inclusion in future DHS and Malaria Indicator Surveys to better characterise traveller groups are included in Additional file The data sets supporting the results of this article are available in Additional file


Plasmodium falciparum malaria, whereas chloroquine is still commonly used for the treatment of non-falciparum species . A more simplified, more uniform treatment approach across all malaria species is worthwhile to be considered both in endemic areas and for malaria as an imported condition alike.Artemisinin combination therapy (ACT) is recommended as first-line treatment for uncomplicated A PROSPERO-registered systematic review to determine the efficacy and safety of ACT for the treatment of non-falciparum malaria was conducted, following PRISMA guidelines. Without language restrictions, Medline/PubMed, Embase, Cochrane Central Register of Controlled Trials, Web of Science, LILACS, Biosis Previews and the African Index Medicus were searched for studies published up to November 2014.P. vivax (n = 35). Five clinical trials in total were identified evaluating ACT for P. ovale, P. malariae and Plasmodium knowlesi. Most ACT presentations have high efficacy against P. vivax parasites; artemisinin-based combinations have shorter parasite and fever clearance times compared to chloroquine. ACT is as effective as chloroquine in preventing recurrent parasitaemia before day 28. Artemisinin-based combinations with long half-lives show significantly fewer recurrent parasitaemia up to day 63. The limited evidence available supports both the use of chloroquine and an ACT for P. ovale and P. malariae. ACT seems to be preferable for optimal treatment of P. knowlesi.The literature search identified 986 reports; 40 publications were found eligible for inclusion, all of them on non-falciparum malaria in endemic areas. Most evidence was available for P. ovale, P. malariae and P. knowlesi should be a research priority.ACT is at least equivalent to chloroquine in effectively treating non-falciparum malaria. These findings may facilitate development of simplified protocols for treating all forms of malaria with ACT, including returning travellers. Obtaining comprehensive efficacy and safety data on ACT use for non-falciparum species particularly for CRD42014009103The online version of this article (doi:10.1186/1475-2875-13-463) contains supplementary material, which is available to authorized users. Plasmodium are known to cause human malaria infections. The majority of cases are caused by Plasmodium falciparum, which can lead to high morbidity and mortality if not treated promptly. Non-falciparum malaria is caused by Plasmodium vivax, Plasmodium ovale subspecies, Plasmodium malariae or Plasmodium knowlesi. Data on the epidemiological patterns of the non-falciparum malarias is scarce. In Asia and the Americas, where malaria transmission is generally low and seasonal, P. falciparum and P. vivax malaria have nearly the same prevalence. Plasmodium malariae and P. ovale are mainly found in sub-Saharan Africa and comprise <10% of isolates. Currently, non-falciparum malaria constitutes approximately 25% of all annually imported 11,000 cases of malaria in Europe[P. knowlesi in endemic areas, whereas chloroquine (CQ) is still the standard drug for P. vivax, P. malariae and P. ovale in most countries[Malaria remains one of the most important and potentially life-threatening parasitic diseases of humans. Five species of the genus in Europe. Artemisin Europe. It is cP. falciparum and non-falciparum malaria may have several drawbacks in clinical practice. Most importantly, misclassification of Plasmodium spp. is common especially where non-falciparum infections are involved, due to lack of training in microscopy and variations in morphological characteristics within and between Plasmodium spp.[P. falciparum can have severe or even fatal consequences due to widespread CQ resistance of P. falciparum. Despite internal and external quality assessments for parasitological diagnosis, discrepant results between microscopy and polymerase chain reaction (PCR) can still occur even in experienced laboratories[P.f/P.v. test based on P. falciparum HRP2/aldolase), are not capable to differentiate safely single species P. falciparum infection from concurrent P. malariae or P. ovale[P. malariae and P. ovale. An evaluation of three rapid diagnostic test  for the detection of P. knowlesi infection demonstrated a low sensitivity  and low specificity and a risk of misdiagnosis of P. falciparum or vivax[The different treatment recommendations for dium spp., 7. CQ toratories. Besidesoratories and microratories, 11. Rap P. ovale. Those t or vivax. AlthougP. vivax resistance to CQ is emerging in parts of sub-Saharan Africa and South East Asia[P. vivax[P. vivax in affected areas[P. ovale and P. malariae to CQ has been reported only once from Indonesia[Secondly, East Asia. A recen[P. vivax. The Worted areas. So far,Indonesia.P. vivax as a major cause of severe malaria and a comparable incidence of severe malaria between P. vivax and P. falciparum was found in infants, children and adults[Finally, intake of CQ is unpopular in many African descendants due to frequently experienced side effects, e.g. pruritus due to Cnd adults.P. falciparum infection, but also offer logistical benefits in terms of pharmacological management, whilst the need to eliminate P.vivax/P.ovale hypnozoites outside endemic areas has to be accounted for. Moreover, using a combination treatment will less likely result in artemisinin resistance development. However, certainty about the efficacy and safety of ACT for non-falciparum malaria and mixed malaria infections is needed before translating ACT treatment for all malaria infections into practice. This review aims at describing the efficacy and safety of ACT for non-falciparum malaria.Hence, there might be a compelling rationale to treat all (including returning travellers) diagnosed with malaria with an ACT irrespective of malaria species. More orThe overall objective of the review was to summarize available data on efficacy and safety of artemisinin combination therapy (ACT) for the treatment of non-falciparum malaria. Primary endpoint was to describe the cure rate at day 28; secondary endpoints were to describe cure rates at days 0–3, 7, 14, 21, 28, 42, 56 and 63, fever clearance time (FCT), parasite clearance time (PCT) and adverse events profiles.This review was conducted in June 2014. The last search was conducted on 16 November 2014. Objectives and inclusion criteria were specified in advance and documented in a protocol. Recommendations made by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) group were followed. This reP. vivax malaria and n = 5 altogether for P. malariae, P. ovale or P. knowlesi contained the necessary data for the qualitative synthesis. The full text was retrievable for all included records except two[The initial search yielded 2,022 records of which 986 remained after removal of duplicates  in patients treated with CQ, compared with 54.9%  in those receiving DHA + PP[P. vivax malaria)[Fifteen randomized clinical trials Table  comparedx malaria. ACT cler than CQ , 48, 49,vs CQ 96%), it seems that P. vivax has developed resistance to pyrimethamine faster than P. falciparum did[P. vivax in many regions.Only one artemisinin combination, namely AS + SP, seems unsuitable for the treatment of vivax malaria. Although one non-inferiority trial, from Afparum did. ConsequTwo trials investigated an ACT versus CQ + SP; one study compared AS + SP with CQ + SP, and oneP. vivax[Six studies, 55, 64 P. vivax, 55, 68.P. vivax, 6,3% mixed) showed no difference in efficacy for treatment of P. vivax or all-species malaria before day 42[max) on day 2. The authors concluded that a compressed 2-day regimen is best avoided until the clinical relevance of these findings are more thoroughly evaluated.An RCT evaluating a two versus three-day regimen of DHA + PP  for uncomplicated malaria  for P. malariae patients treated with artesunate versus no cure (95% CI 0-15%) in the placebo group. Treatment with artesunate led to a parasitological cure rate of 83% by day 56 in this semi-immune population. However, there may have been possible re-infections during the 56-day follow-up period. The true cure rate is likely to be underestimated as these results were not corrected for by PCR. In another clinical trial, artesunate was 100% effective for the treatment of P. ovale infections, with a PCT and FCT of < 48 hours[P. ovale) and no anti-relapse therapy with primaquine administered for P. ovale. One other case report is available, which reported good efficacy of intravenous artesunate for mixed P. falciparum/P. malariae infection[P. falciparum/P. malariae infection in a HIV-infected male and ineffectiveness of artemether/lumefantrine to clear the infection of P. malariae[P. malariae or a natural induced drug resistance of the parasite, which have never been published before in quartan malaria. However, single case reports leave open questions, and hypotheses on lack of ACT efficacy against P. malariae remain to be somewhat weak.Epidemiology of nt review and briedentified, 61, 62.infection. This st malariae. This coP. knowlesi malaria is provided in a recent review[P. knowlesi malaria, and intravenous artesunate for severe knowlesi malaria[P. knowlesi infections in humans[P. vivax or P. knowlesi), was two days, with 55/119 (42%) of P. knowlesi infected patients being smear-negative by day 1 (P = 0.032). The median fever clearance time was one day in P. knowlesi compared to two days for P. falciparum malaria. Twenty-three (18%) P. knowlesi patients were followed up during days 26–41, with no recrudescence identified. For severe knowlesi malaria, intravenous artesunate was successfully used. In this study, >80% (15 of 18) of the patients with a P. knowlesi parasitaemia >100,000 parasites/μL met other severity criteria, supporting the use of this cut-off value for mandating intravenous artesunate. William and colleagues demonstrated excellent efficacy of AL (n = 8) for uncomplicated knowlesi malaria in a retrospective case review study[P. knowlesi malaria, 16 were treated with intravenous quinine, six received intravenous artesunate. The median PCT was significantly shorter with artesunate  than with quinine  (p = 0.02). Six patients died, of which 5 had been administered quinine , and 1 was given artesunate . Only a few others report on the treatment of P. knowlesi with ACT. A patient from Pahang Province  was unsuccessfully treated with mefloquine, but recovered with intravenous quinine in combination with artemether-lumefantrine[P. knowlesi infection in a traveller from Thailand in Germany. Parenteral treatment was started (2.4 mg/kg) and the patient was switched to oral AL the next day, on which she fully recovered[A detailed overview on the history, clinical symptoms and diagnosis of nt review. Becausent review do not pnt review in theiri malaria. A recenin humans. The meaiew study. The mediew study. Of the efantrine. Anotherrecovered.P. falciparum malaria is extensive, and rapidly growing for P. vivax malaria. Clinical trials of ACT for P. malariae, P. ovale and P. knowlesi are much scarcer and limited to non-randomized studies, case-series or case-reports.Evidence for the efficacy of ACTs for P. vivax parasites, both sensitive and resistant to CQ. Studies identified in this review showed good efficacy of ACT against the blood stages of P. vivax, with quicker parasite and fever clearance times compared to CQ monotherapy. Countries with CQ-resistant P. vivax are already implementing a unified ACT-based treatment policy[P. vivax is, after the first report in 1989 from Papua New Guinea[P. vivax malaria treatment efficacy studies to establish the global extent of chloroquine resistance[P. vivax. Also, within countries close to elimination of malaria, an increasing proportion of P. vivax malaria is imported and, outside sub-Saharan Africa, the proportions of all cases caused by P. vivax are rising[P. vivax to non-endemic areas, the best treatment remains to be elucidated; in these cases, the economic, malariometric, and operational costs of ACT versus CQ need to be weighted by the clinician. Different regimens are still acceptable if diagnostic tests reliably differentiate CQ-resistant P. falciparum from P. vivax. However, this is even in experienced laboratories not always the case. Moreover, the paradigm among infectious diseases experts that bacterial infections should be treated as “narrow as possible” does not apply to treating malarial infections. Monotherapy for malaria will only increase the likelihood of resistance, in contrast with narrowing down antibiotic therapy resulting in lower levels of bacterial resistance[Several artemisinin-based combinations yield high efficacies against nt policy, 78. Incew Guinea, reporteew Guinea–83, Myanew Guinea, Papua New Guinea, Vietnamew Guinea, Turkey[ew Guinea, 88, Colew Guinea, Braziliew Guinea and Ethiew Guinea, 92–94. esistance. Althougre rising. In travesistance.P. vivax malaria (NCT02110784).A position paper (2012) from the European Society for Clinical Microbiology and Infectious Diseases, summarized main issues regarding the management of imported malaria. For theP. vivax, has been jeopardized by the supposedly higher economic costs[So far, the use of ACT for non-falciparum malaria, in particular mic costs. This juP. falciparum and non-falciparum strains[The use of ACT for non-complicated non-falciparum malaria infection is convenient but may imply the risks’ of a higher utilization of the combination drug, with two possible drawbacks. Firstly, the availability of good quality drugs may be limited in malaria endemic countries; howeverm strains.P. falciparum malaria, P. vivax, and to a lesser extent P. ovale, form hypnozoites, which remain dormant in liver cells after an acute infection. Therefore, in P. vivax infections  the removal of blood schizonts is not sufficient to clear the infection, as the eradication of hypnozoites  is required to prevent relapses. This is clinically important and cost-effective particularly in travellers who are not likely to be re-infected.In contrast to P. vivax recurrent parasitaemia. However, neither ACT nor CQ is active against P. vivax hypnozoites and thus the treatment must be combined with primaquine, currently the only approved drug able to achieve radical cure. WHO guidelines recommend a course of primaquine for the radical treatment of P. vivax of at least 14 days[P. vivax and P. ovale. The assessment of the prophylactic activity and pharmacokinetic profile of oral tafenoquine compared to primaquine for inhibition of liver stage malaria infections demonstrated a much improved efficacy of tafenoquine when compared to primaquine[P. vivax malaria relapse prevention was more efficacious than CQ alone, with a similar safety profile[ACT does not affect the hypnozoites in the liver, but ACT with a long half-live partner drug, such as piperaquine 23–28 days,  for P. ovale and P. malariae. It is advisable to treat with ACT if species classification is not 100% certain, because P. ovale and P. malariae often occur in mixed infections with P. falciparum, which is likely to be CQ-resistant[in vivo testing; in vitro and in silico methods to assess drug resistance of non-falciparum species remain very much limited up to date. P. ovale and P. malariae are considered sensitive to CQ. However, there is one report on CQ-resistant P. malariae from South Sumatra, Indonesia[P. malariae, P. ovale curtisi and P. ovale wallikeri was observed. This apparent “treatment failure” requires more investigation. It is important to note, however, that in vivo anti-malarial drug efficacy cannot really be determined from data derived from outcomes of asymptomatic infections. Furthermore, their method of measurement of outcome, PCR, is not yet validated as a primary endpoint method for anti-malarial drug trials. It is speculated by the authors that a relapse of P. ovale could explain the persistent detection of parasites; however, the prophylactic effect of piperaquine which is generally assumed to last more than three weeks makes this less plausible[P. malariae and P. ovale were detected by PCR, and not the asexual stages, as it is known for P. falciparum that gametocyte carriage following a dihydroartemisinin-piperaquine regimen is more frequent than for other artemisinin-based combinations (e.g. AL)[P. malariae, the long intra-erythrocytic cycle of three days may explain their persistence due to dihydroartemisinin short Tmax (±2 h) and rapid clearance by CYP3A4/5 in the liver, and, therefore, may be more likely to survive after each dose of artemisinin. Similar observations have been made concerning delayed parasite clearance times in P. malariae in Madagascar, where a patient treated with CQ was still positive for P. malariae (detected by PCR) on day 7, although it was subsequently cleared with no late recurrence[P. malariae, showed a significant lower clinical response rate, with nearly 20% of patients still parasitaemic by day 2 and one patient remaining parasitaemic on day 4[P. ovale and P. malariae mono-infections are difficult to realize due to patient’s safety issues; misdiagnosis of P. falciparum malaria can have fatal consequences.At present, al trials, 61, 62 m malaria. More evresistant. KeepingIndonesia. SimultaIndonesia. Of concIndonesia. A persiplausible. Further(e.g. AL). In P. mecurrence. Another on day 4. CQ’s ma on day 4. AlthougP. knowlesi are lacking, several studies have demonstrated the efficacy and safety of ACT (particularly AL)[P. knowlesi malaria, it is recommended, in view of the potential severity and velocity at which the disease can develop, to use an ACT that is suited for the treatment of P. falciparum or P. malariae for uncomplicated P. knowlesi malaria. This is particularly relevant for elderly patients because they are at greater risk of severe disease due to the strong correlation between age and parasite count[Although currently randomized clinical trials on the treatment of larly AL), 66. Altite count, 111. Thite count. The impite count and rapiite count. Parenteite count as it isP. malariae infection harboured P. knowlesi; approximately 10% were actually infected with P. falciparum[P. knowlesi endemic areas, with a high prevalence of CQ-resistant falciparum malaria, negligent use of CQ for misdiagnosed P. falciparum malaria may have detrimental consequences. Also, resistance of P. knowlesi to anti-malarial drugs have not been observed yet[Plasmodium knowlesi was responsible for almost half of the malaria deaths in Sabah (Borneo), due to misdiagnosis with P. malariae and delayed administration of artesunate for severe non-falciparum malaria[In studies conducted in Sarawak, it was demonstrated that whilst most patients with microscopy-diagnosed alciparum. In traverved yet. Moreoveerved yet. Plasmodm malaria. In a rem malaria. More trm malaria), a RCT P. vivax) in endemic areas and among travellers, the clear recognition as non-benign infection and the emergence of CQ-resistant strains call for a paradigm change in the way non-falciparum malaria infections are treated. Although non-falciparum malaria remains susceptible to CQ in parts of the world, the role of ACT in the treatment of non-falciparum malaria in endemic as well as non-endemic areas should be acknowledged. ‘Even’ if most non-falciparum infections do not take a potentially life-threatening course, the shorter parasite and fever clearance times alone make a strong case for ACT following establishment of the correct diagnosis. A more uniform treatment for malaria would offer important organizational, logistical and economic advantages, next to malariologic advantages of ACT in preventing resistance, showing rapid parasite and fever clearance times and good cure rates. However, the incomplete nature of current data on the efficacy of ACT in the treatment of P. ovale, P. malariae and P. knowlesi in endemic areas but also as imported conditions compels current malaria researchers to evaluate the efficacy, safety and cost-effectiveness of these drugs in randomized controlled clinical trials. Because most studies have been carried out in endemic settings, one must be careful transferring these results directly to non-immune travellers with imported non-falciparum malaria. On the other hand, European treatment guidelines are mainly based on data from studies carried out in endemic areas, since there is a paucity of original prospective treatment data for non-immune patients[The considerable burden of non-falciparum malaria (predominantly  patients. It must patients.This documents describes the full methods of the present review.(DOC 33 KB)Additional file 1:This PDF file depicts tables with the exact search strategy.(PDF 56 KB)Additional file 2:This document provides basic background information onP. ovale,P. malariaeandP. knowlesiinfections.(DOC 178 KB)Additional file 3:


Plasmodium falciparum histidine-rich protein 2 (pfhrp2) gene that may compromise the use of pfhrp2-based rapid diagnostic tests (RDTs) for the diagnosis of malaria was assessed in P. falciparum isolates from Yemen.The genetic variation in the CareStart™ malaria HRP2-RDT and Giemsa-stained thin and thick blood films. The Pfhrp2 gene was amplified and sequenced from 180 isolates, and subjected to amino acid repeat types analysis.This study was conducted in Hodeidah and Al-Mahwit governorates, Yemen. A total of 622 individuals with fever were examined for malaria by P. falciparum by the RDT. Overall, 12 different amino acid repeat types were identified in Yemeni isolates. Six repeat types were detected in all the isolates (100 %) namely types 1, 2, 6, 7, 10 and 12 while types 9 and 11 were not detected in any of the isolates. Moreover, the sensitivity and specificity of the used PfHRP2-based RDTs were high .A total of 188 (30.2 %) participants were found positive for pfhrp2 gene, and its potential impact on the PfHRP2-based RDTs commonly used in Yemen. CareStart™ Malaria HRP2-based RDT showed high sensitivity and specificity in endemic areas of Yemen.The present study provides data on the genetic variation within the The online version of this article (doi:10.1186/s13071-015-1008-x) contains supplementary material, which is available to authorized users. Plasmodium falciparum is the predominant species and was responsible for almost 99 % of malaria cases in Yemen during 2012, a large number of which consisted of drug-resistant P. falciparum parasites [Malaria is still a major public health problem in Yemen, with almost 66 % of the population living in areas that suffer from stable malaria transmission . Plasmodarasites . Among 1arasites .The national malaria control programme in Yemen (NMCP) has achieved substantial success in controlling local cases of malaria, achieving a significant reduction in the number of malaria cases, dropping from 900,000 cases in the early 2000s to around 150,000 cases by 2013 [P. falciparum malaria [Early and accurate diagnosis of malaria, along with prompt treatment, are essential to reduce the burden of the disease worldwide. Rapid diagnostic tests (RDTs) have been widely used for the diagnosis of malaria and become an indispensable tool for malaria case-management, control and elimination worldwide, especially in rural endemic areas without laboratory access , 7. Besi malaria –11.pfhrp2 are not available. Hence, the present study aims to investigate the genetic variations of the pfhrp2 gene in malaria isolates from the Hodeidah and Al-Mahwit governorates, Yemen  and the possible impact of this variation on the efficacy of the currently used pfhrp2-based RDTs. The present study is the first to provide data on the genetic variation of PfHRP2 in Yemen, and therefore has the potential to impact on control strategies and efforts to eradicate malaria from Yemen and the Arabian Peninsula.In Yemen, PfHRP2-based RDTs have been implemented by the NMCP in 2009, and are being used exclusively for malaria active case detection (ACD) targeting only falciparum malaria infections . HoweverAn active case detection survey targeting individuals with fever suspected of having a malaria infection was carried out in some malaria endemic districts of the Tehama region in both the Hodeidah and Al-Mahwit governorates, Yemen. The survey was carried out from March to May 2014, during the malaria transmission season. Districts with high malaria endemicity; namely AdDahi, Al-Marawiah, and Bajil from Hodeidah, and Khamis Bani Saad from Al-Mahwit, were selected based on the national malaria records of 2010–2013 provided by NMCP.2 with a total population of 2.16 million [2 with a total population of 597,000 people [The Hodeidah governorate  is located in the Tehama region in the western part of Yemen, about 226 km from Sana’a, the capital of Yemen. It is a coastal area located along the Red Sea, covering a total area of 117,145 km million . Al-Mahw0 people . The cliCareStart™ Malaria HRP2, Cat no. G0141, Access Bio, Inc, USA), and for preparing both thick and thin blood films. Filter paper blood spots were also collected from each participant on 3MM Whatman® filter paper , and kept in clean, dry, well-sealed aluminum pouches with desiccated silicon bags for molecular analysis. Blood films were stained with 5 % of buffer-diluted Giemsa stain for 30 min and were examined microscopically for the presence of malaria parasites. All RDT tests were performed and interpreted by trained and skilled laboratory personnel from the NMCP following the manufacturer’s instructions. Briefly, one purple line in the control line position interpreted negative for falciparum malaria and two purple lines at the test line position along with the control line were to be defined as positive. In case the control line did not appear, the result was considered invalid and the test was repeated.A total of 622 individuals with fever were recruited to this study and examined for malaria. Finger prick blood samples were collected from participants and tested using the RDT  was determined by counting only the asexual stages against 300 white blood cells (WBC) and then multiplied by 25; assuming the average of total WBC count of individuals equal to 7500 cells per μl of blood. The level of parasitaemia was graded as low (<1000 parasites/μl of blood), moderate (1000–9999 parasites/μl of blood) and severe . A double check for malaria microscopy was performed by two senior malaria microscopists; slides were examined twice and the average parasitemia per microlitre of blood was recorded for every microscopy positive slide. Genomic DNA was extracted from the filter paper blood spots and subjected to The protocol used in this study was approved by the Ethics Committee of the University of Malaya Medical Centre, Malaysia (Ref. 974.19). The protocol was also approved by the Ministry of Health and Population, in conjunction with the National Malaria Control Programme in Yemen. Written and signed or thumb-printed informed consents were taken from adult participants and parents or guardians on behalf of their children before starting the sample collection; these procedures were also approved by the ethics committees. RDT-positive participants were treated with artemisinin combination therapy (artemisinin + sulfadoxine/pyrimethamine) according to the national malaria treatment policy, Ministry of Health and Population, Yemen.One or two discs (6 mm diameter) of 3MM Whatman’s filter paper blood spot (cut by flamed-sterile punch) were used for DNA extraction using a Qiagen blood and tissue kit  according to the manufacturer’s instruction. DNA was eluted using 50 μl AE  elution buffer (included in the kit) and kept at −20 °C until used.P. falciparum mono-infection, was chosen to evaluate the CareStart™ Malaria HRP2-RDT. The blood sample was also checked not to have any sexual stage (gametocytes). Duplicate thick and thin blood films were prepared and stained with Giemsa stain as previously mentioned, and then examined microscopically by two senior malaria microscopists; the average of parasitaemia was recorded. The sample was then diluted in a ratio of 2:3 serial dilutions with healthy human O positive blood group. A total of 12 serial dilutions (labelled 1–12) were prepared in duplicates and the results of asexual-stage parasites density were recorded for each tube [A malaria-positive blood sample, with a known parasite density  of ach tube . Each tuP. falciparum samples then being considered for pfhrp2 molecular characterization. Amplification of hrp2 was carried out in a single-run PCR using a specifically designed oligonucleotide primer pair flanking the region of exon-2 of pfhrp2 gene [P. falciparum lines 3D7 (MRA-102G), Dd2 (MRA-150G) and HB3 (MRA-155G) provided by Malaria Research and Reference Reagent Resource Centre (MR4), ATCC®, Manassas, VA, USA were used as positive and negative controls for PCR amplification of HRP2 and HRP3. 3D7 was used as a positive control for both HRP2 and HRP3, and Dd2 (a laboratory line known to lack pfhrp2) and HB3 (a laboratory line known to lack pfhrp3) were used as negative controls for HRP2 and HRP3 respectively [All DNA from malaria positive samples were confirmed by PCR , with P.rp2 gene . A 50 μLectively , 18.The PCR products were then analysed using agarose-gel electrophoresis. Ten microliters of each amplicon was loaded into a 1.5 % agarose gel and run in a TAE buffer (Tris acetate EDTA), stained with SYBR® safe DNA gel stain . The fragments size was visualized under UV compared to 100 bp DNA ladder.The amplicons were sent for purification and sequencing, each amplicon was subjected to sequencing using the same forward and reverse primers as were used during PCR amplification. Both forward and reverse sequences were aligned using BioEdit Sequence Alignment Editor Software (version 7.1.9) and then translated into corresponding amino acids. Each sequence of amino acid repeats was identified and given a code from Type 1 to Type 14 based on the motif being repeated .P-value of < 0.05 was considered significant.The performance of RDT was calculated based on the following indicators: sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV) which were calculated with their corresponding 95 % confidence intervals (CI) using Medcalc® online calculator. Moreover, Kappa statistics were used to assess the agreement between RDT and microscopy. This was calculated in IBM SPSS Statistics, version 18.0  by creating a 2 × 2 contingency table. A CareStart™ Malaria HRP2-RDT. Of these 188 RDT-positive, only 171 (91.0 %) were confirmed microscopically by detecting either asexual or sexual stages or both. On the other hand, 18 (2.9 %) microscopy-positive individuals were found to be negative with RDT. Similarly, 17 (2.7 %) RDT-positive cases were found to be negative with microscopy. Based on microscopy, the detection rate was the highest in Khamis Bani Saad (39.4 %) followed by Bajil (23.5 %), while Al-Marawiah has the lowest (5.3 %). The level of asexual P. falciparum parasitaemia in positive cases ranged from 40 to 55,555 parasites/μl of blood with a geometric mean of 5261 parasites/μl. High parasitaemic individuals  represented 17.9 %, while 41.9 % and 40.2 % of the malaria-positive individuals had moderate and low parasitaemia respectively.Of the 622 screened individuals, 188 (30.2 %) were positive for malaria by the CareStart™ malaria HRP2-RDT was rated as 90.5 %  and 96.1 % , respectively (Table P < 0.001). In the same vein, HRP2-RDT was examined against serial dilutions of a blood sample with known P. falciparum parasite density. The test revealed a high detection rate of CareStart™ malaria HRP2-RDT for falciparum malaria isolates; 10 out of the 12 tubes with parasitaemia levels ranged from 8500 to 147 asexual stage/μl were found positive (visible clear band), with a faint band appearing for tube no.11 (98 parasites/μl). The last tube, no. 12, with parasitaemia of 65 parasites/μl was found negative by HRP2-RDT , with a sequence of 540 bp (180 amino acids) being the most frequent genotype (43.9 %). All isolates sequences were found to be similar in that they started with either a single or multiple Type 1 (AHHAHHVAD), and ended with a single Type 12 repeat (AHHAAAHHEAATH). Overall, Type 1 (AHHAHHVAD), Type 2 (AHHAHHAAD), Type 6 (AHHATD), Type 7 (AHHAAD), Type 10 (AHHAAAHHATD) and Type 12 (AHHAAAHHEAATH) were found in all sequenced isolates. On the other hand, type 9 (AAY) and Type 11 (AHN) were totally absent in all isolates collected from both governorates.Overall, 180/189 cases were successfully amplified for pfhrp2 were the highest among the 12 types detected by this study . No association was found between HRP2 repeats and age, sex and districts of participants as well as parasitaemia level. The frequency and mean number of PfHRP2 repeat types from P. falciparum isolates from Hodeidah and Al-Mahwit, Yemen are shown in Table Other amino acid repeat types were varied in their frequencies. Type 8 (AHHAAY), Type 5 (AHHAHHASD) and Type 4 (AHH) ranged between moderate and low, with percentages of 49.4 %, 47.2 % and 15.6 % respectively. Moreover, Type 13 (AHHASD) and Type 14 (AHHAHHATD) were rarely reported in the present study . Further, the mean numbers for 2 and 7 repeat types in CareStart™ malaria HRP2-RDT and microscopy but it was PCR-negative for pfhrp2 gene. DNA of this isolate was processed for further PCR confirmation using three PCR protocols that aimed at amplifying P. falciparum 18SrRNA [pfhrp2 and pfhrp3 genes [18SrRNA and pfhrp3 genes while it was confirmed negative for pfhrp2 gene.Interestingly, one isolate with low parasitaemia (184 parasites/μl) was found to be positive by both  18SrRNA , pfhrp2 p3 genes . The DNAP. falciparum specific proteins; either histidine-rich protein 2 (PfHRP2) or P. falciparum lactate dehydrogenase (PfLDH), while others can recognize both P. falciparum-specific and pan-specific antigens  and pan-species (pLDH) [P. falciparum is a 2-exons gene connected with an intron, located on chromosome 7 (98671–99734 bp) [pfhrp2 have been characterized into 14 types, based on amino acid motifs being repeated, with Type 2 and Type 7 having been described as possible epitopes targeted by the monoclonal antibodies used to detect hrp2 [Many rapid diagnostic tests (RDTs) are available to detect different malaria-specific antigens. Most RDTs detect s (pLDH) . Previous (pLDH) , 22. The9734 bp) . The amiect hrp2 , 21, 24pfhrp2 for the Yemeni isolates were found to be lower than the global variation (187 to 306 amino acids), as reported for isolates from 19 countries in Africa, South America, the Pacific region and Southeast Asia [The present study investigated the genetic variation of PfHRP2 among isolates from Yemen, with a possible predicting effect on the performance of the PfHRP2-based RDTs  that are used solely for active case detection (ACD) by the national malaria control programme in Yemen (NMCP). A total of 180 isolates were successfully amplified and sequenced for PfHRP2. The sequence lengths of the sequenced isolates varied from 477 to 879 bp (giving proteins of 159 to 293 amino acids). The variations in the number of total amino acids for P. falciparum isolates [Overall, 12 different amino acid repeat types were identified in Yemeni isolates examined by the present study. Of these, six repeat types  were found to be present in all isolates, while type 3 was found in 98.3 % of the isolates. These findings are in agreement with isolates from different countries in Africa, Asia and America . In Indiisolates . On the isolates . Our stuisolates , 16.pfhrp2 sequences begin with a type 1 repeat and concluded with a type 12 repeat [P. falciparum isolates had detected three isolates for which pfhrp2 protein sequences did not begin with a type 1 repeat, and only one isolate found in that study possessed a sequence ending in a type 12 repeat [We found that all of the amplified isolates 100 %) started with a type 1 repeat (AHHAHHVAD), with either a single or multiple of 2 to 5 copies, before ending with a single Type 12 repeat (AHHAAAHHEAATH). Many previous studies in Africa and Asia showed that all 2 repeat , 19, 25.0 % startP. falciparum isolates [pfhrp2 vary considerably between countries and within the same country [pfhrp2  which is higher than what it has been reported in Thailand, the Philippines, Madagascar, Papua New Guinea and South America [The present study also showed that the existence of other repeat types in Yemeni isolates varied from moderate  to low . Moreover, other types were found only rarely . Our findings are consistent with a previous report on Senegalese isolates . By contisolates . Furtherisolates . Moreove country , 19. Yem America , 19, 26.P. falciparum at a parasitaemia greater than 500 parasites/μl, with most of the variation reported at relatively low-level parasitaemia [Our findings revealed that the HRP2-based RDT possessed good sensitivity; a low parasitaemia level of 139 asexual stages/μl was strongly detectable by RDT. On the other hand, parasitaemia levels below 100 parasites/μl were found either weakly detectable (98 parasites/μl) or totally not detected (65 parasites/μl). These results correspond to RDT results on the Yemeni isolates as we found that detection rate increased with parasitaemia level. However, low detection rate (44.4 %) was reported with samples of very low parasitaemia , as well as two samples (positive with microscopy and pfhrp2-PCR) with moderate and high parasitaemia were found negative. These are in agreement with previous studies which revealed that most of the RDT show excellent detection rates for sitaemia , 10, 21.sitaemia , 28. Thisitaemia , 19, 29.Overall, the present study showed that the sensitivity and specificity of the used HRP2-based RDT were high . This was supported by a very good level of agreement between the results of microscopy and RDT tests (Kappa = 0.867). The sensitivity of PfHRP2-based RDT from other reports varied worldwide, ranging from > 90 % (high) to 43.7 % (low) compared to the gold standard microscopy and/or PCR –34. With18S rRNA and pfhrp3 genes. Moreover, the HRP2 PCR protocol was tested against falciparum malaria DNA samples with the same and lower parasite densities which all yielded successful amplification for HRP2 gene suggesting PfHRP2 gene deletion. Due to pfhrp3 and pfhrp2 structural homology, pfhrp3 can cross-react with HRP2-coated antibodies in the RDT [pfhrp2 gene deletion was reported worldwide and more extensively from South America. It was first reported among Peruvian isolates; 41 % of the malaria-microscopically positive isolates have been found negative by RDT, and failed to amplify the PfHRP2 gene by PCR [pfhrp2 gene deletion [In the present study, one sample was found positive for falciparum malaria by microscopy and HRP2-RDT while it was negative by PfHRP2-PCR. The DNA quality was confirmed by successful PCR amplification of both falciparum  the RDT , and thie by PCR . Later, e by PCR , 37, 38,e by PCR , and frodeletion .pfhrp2 in P. falciparum isolates from Yemen, and reveal that pfhrp2 is highly polymorphic in these isolates. CareStart™ one-step HRP2-based RDT showed high performance especially for those cases with parasitaemia of >100 parasites/μl. The isolates used by this study were from endemic areas of the Tehama region only. Therefore, population-based studies from other endemic areas throughout Yemen are recommended in order to genetically analyze the P. falciparum isolates based on PfHRP2, and to accurately determine the prevalence of parasites that cannot be detected using the PfHRP2 RDT.The findings of this study provide insights into the genetic diversity of


Placental malaria (PM) causes adverse pregnancy outcomes in the mother and her foetus. It is difficult to study PM directly in humans due to ethical challenges. This study set out to bridge this gap by determining the outcome of PM in non-immune baboons in order to develop a non-human primate model for the disease.Plasmodium knowlesi blood-stage parasites through the femoral vein on 160th day of gestation (for pregnant baboons). Peripheral smear, placental smear, haematological samples, and histological samples were collected during the study period. Median values of clinical and haematological changes were analysed using Kruskal-Wallis and Dunn’s Multiple Comparison Test. Parasitaemia profiles were analysed using Mann Whitney U test. A Spearman’s rank correlation was run to determine the relationship between the different variables of severity scores. Probability values of P <0.05 were considered significant.Ten pregnant baboons were acquired late in their third trimester (day 150) and randomly grouped as seven infected and three non-infected. Another group of four nulligravidae (non-pregnant) infected was also included in the analysis of clinical outcome. Malaria infection was intravenously initiated by s = 0.7650, P <0.05) and inflammatory score . Although the sample size was small, absence of parasitized erythrocytes in cord blood and foetal placental region suggested lack of congenital malaria in non-immune baboons.Levels of white blood cells increased significantly in pregnant infected (34%) than in nulligravidae infected baboons (8%). Placental parasitaemia levels was on average 19-fold higher than peripheral parasitaemia in the same animal. Infiltration of parasitized erythrocytes and inflammatory cells were also observed in baboon placenta. Malaria parasite score increased with increase in total placental damage score  of baboons that are non-immune to malaria. This is a key feature of placental falciparum malaria in humans. This presents the baboon as a new model for the characterization of malaria during pregnancy. Plasmodium falciparum causes up to 10,000 cases of malaria-related deaths in pregnancy, mainly due to maternal anaemia, and approximately 200,000 infant deaths annually [Every year, 125 million women are exposed to the risk of malaria worldwide [annually . PM leadannually ,5. This annually .Plasmodium knowlesi-infected baboons. Results from this study will contribute to the validation of the baboon-P. knowlesi model of malaria in pregnancy.Over the years, substantial efforts have been made to prevent and control malaria by employing different strategies ,6,7. ThePapio anubis) weighing between 12 Kg and 23 Kg were acquired from the animal facility of Institute of Primate Research (IPR), Kenya, and housed in individual squeeze-back cages (dimensions 0.6×0.6×0.68 m) in the biocontainment facility according to institutional standards. The animals were fed on a standard non-human primate diet, water provided ad libitum and their general health monitored throughout the experimental period [Fourteen female adult baboons  clone blood stage parasites [th day of their gestation. Prior to infection, cryopreserved P. knowlesi parasites were retrieved and cultured overnight by modification of Rowe et al. method [th day of gestation for non-infected control baboons, in order to collect intact placentas. Oral treatment with pyrimethamine (1 mg/kg of body weight for three days) was initiated after CS for the pregnant animals and at peak parasitaemia for nulligravidae.Malaria infection was initiated with 2×10arasites ,19. Four. method ,20. Paraet al. [et al. [Peripheral blood samples were collected for haematological analysis at three intervals, i.e., baseline (before infection), after infection and after treatment, respectively. Intact placentas were harvested during CS according to standard operating procedures and the delivered infants taken to the nursery soon after birth. Placental and umbilical cord blood was collected according to Brustoski et al.  and Moor [et al. , respectet al. [In order to assess the progression of the disease, baboons were monitored for parasitaemia and clinical symptoms, such as fever, appetite, lethargy, and weight changes. Peripheral parasitaemia was determined daily by finger-prick method from day 2 post infection. To determine placental and cord parasitaemia, blood samples collected in capillary tubes were used ,22. Thinet al. .et al. [Venous blood was collected in heparin tubes and haemoglobin (Hb), red blood cells (RBC) and white blood cells (WBC) counts determined at baseline, after infection and after treatment. Gross pathological examination was done during CS by a veterinary pathologist. Placentas were observed for any form of extensive tearing, damage or gross abnormalities. Several placental biopsy specimens of approximately 2×2 cm were obtained for each placental sample, fixed in 10% neutral buffered formalin and later stained with haematoxylin-eosin (H&E). The stained tissues were examined according to Davison et al.  with a fData was analysed using Graph Pad Prism (version 5.0). Experimental animals were grouped as pregnant infected, nulligravidae infected and pregnant not infected. Median values of clinical and haematological changes of these groups were compared using Kruskal-Wallis statistical test and further analysed using Dunn’s multiple comparison test. Parasitaemia profiles were analysed using Mann Whitney U test. A Spearman’s rank correlation was run to determine the relationship between the different variables of severity scores. Probability values of P <0.05 were considered significant.This study was carried out under the Malaria Research Programme at the Institute of Primate Research (IPR) located in Karen, Nairobi, Kenya. The Institutional Review Committee (IRC) comprising animal care and use committee (ACUC), scientific review and the research ethics and integrity team approved all protocols and use of animals in this study.In this study, malaria patency was observed on days 2 and 4 post-infection (PI) in nulligravidae and pregnant infected baboons, respectively. Although peak peripheral parasitaemia (at day 8 PI) was on average 50% higher in nulligravids compared to pregnant baboons, this change was not significant (P = 0.4439) Figure . ChangesThe harvested dams were maroon-red in colour with intact membranes. The maternal surface of the placenta had cotyledons while the foetal surface was densely distributed with blood vessels. Baboon placentas weighed on average 167.3 g and 165.9 g for the infected and non-infected groups, respectively. All animals had no fibrinoids except one (pan3233), which had slightly marked fibrinoids. Slight placental calcification was observed in all groups. Cord insertion was intact either centrally or eccentrically placed and with three blood vessels regardless of the group. Placental praevia was complete and no haemorrhage, whether retro-placental or retro-membranous was observed in all the examined placentas. These observed features present similarity of baboon placenta to human placenta.Thin blood smears prepared from placental maternal surface, foetal surface, cord blood, and maternal peripheral circulation were observed and compared for parasitaemia levels. Placental parasitaemia  was on average 19-fold higher than peripheral parasitaemia in the same animal. This difference was significant (P = 0.00823). Parasitaemia differential count demonstrated the abundance of rings and late stage parasites (schizonts) in peripheral and placental  blood samples respectively Figure  and 5. Iet al. [s = 0.7650, P <0.05) and inflammatory score . This study demonstrated that placental damage and infiltration of immune cells was directly associated with P. knowlesi infection in baboons and subsequent accumulation in the placenta.H&E-stained placental biopsies were analysed by severity score ranging from 0 to 4, with 0 representing none, 1 representing minimal and 4 representing the most severe according to Davison et al.  with a fP. knowlesi infection in pregnancy, symptomatic disease is manifested in combination with increased placental parasitaemia. Consequently, accumulation of parasites in the placental IVS combined with infiltration of inflammatory cells leads to placental damage.This study demonstrated, for the first time, the infiltration of parasitized RBC and inflammatory cells in the placentas of non-immune baboons. During In areas of stable malaria transmission, asymptomatic infections in pregnant women are common, with low clinical malaria presentation due to acquired immunity over time. In contrast, pregnant women in unstable malaria transmission areas, such as the Asia-Pacific region and South America present clinical disease because of low levels of acquired immunity . This stP. falciparum [falciparum malaria [P. knowlesi-infected non-immune baboons.Placental falciparum malaria in humans increases neonatal mortality by lowering birth weight while fever is associated with premature birth . Increaslciparum . It is e malaria ,32 and u malaria ,34. Seve malaria ,36. Redu malaria . A similP. knowlesi-infected baboon placentas were observed in this study. Immunosuppression during pregnancy is important in maintaining the foetal allograft. Although some murine and human studies have demonstrated that suppression of cell-mediated immune responses plays a major role in the increased susceptibility to malaria during pregnancy [Plasmodium falciparum hyperendemic area in Tanzania revealed a marked increase in the levels of monocytes and macrophages and cytotoxic T cells in the IVS of placentas with active malaria infection [Increased levels of WBC in peripheral circulation and inflammatory cell  infiltration in H&E-stained tissues obtained from regnancy ,25,38-40nfection . FindingRetroplacental transmission of falciparum malaria parasites from mother to her foetus is rare in humans particularly in CS deliveries. For instance, a study conducted in Burkina Faso, a region of stable malaria transmission, malaria rate in umbilical cord blood occurred in 1.4% of all newborns. This mechanism of congenital transmission was not clear, although it was attributed to several factors ,43. HoweP. knowlesi parasites in the baboon placenta. It is documented that only the mature falciparum malaria parasites show cytoadherence properties [P. falciparum erythrocyte membrane protein 1 (PfEMP-1), a large, highly variant parasite antigen protein, encoded by the var multigene family [P. knowlesi infected erythrocytes in the baboon placenta, chondroitin sulphate proteoglycan (CSPG) 4 and HAPLN 1 were predicted as putative receptor molecules in the baboon with high similarity to human CSA and HA respectively. In addition, P. knowlesi erythrocyte binding proteins  matched closely to the placental P. falciparum ligand Var2csa [P. knowlesi parasites in baboon placenta.This study has also demonstrated accumulation of parasitized erythrocytes in the IVS of the baboon placenta, in addition to high levels of mature forms of the parasite in the placenta. This suggests possible cytoadherence of operties . These poperties . In factoperties ,46. The e family . The adhe family -50. In s Var2csa . FurtherP. knowlesi associated PM in the baboon.Histopathological findings demonstrated that PM in baboons is characterized by placental damage due to fibrinous necrosis of the villi, chorionic plate thrombosis, syncytiotrophoblast disruption, chorionic plate syncytiotrophoblast disruption, and thickening of trophoblastic basement membrane. The process was also accompanied with infiltration of inflammatory cells in the placental tissue was. These findings correlate well with human studies where placental falciparum malaria pathology is characterized by excess perivillous fibrinoid deposits, excessive syncytial knotting, trophoblastic membrane thickening, which have been associated with destruction/damage of placental tissues, and proliferation of cytotrophoblastic cells ,25,46,52knowlesi PM in baboons mimic placental falciparum malaria in humans. Compared to other non-human primates used previously, the P. knowlesi-baboon model has demonstrated accumulation of parasitized erythrocytes in the placental IVS, a key feature of placental falciparum malaria in humans. Therefore, this study presents the new baboon model for characterization of malaria in pregnancy. This will, for example, be relevant in understanding host molecular mechanisms involved in sequestration, a key determinant of pathogenesis associated with falciparum and knowlesi malaria in pregnancy.Pathological and clinical features of


Cotrimoxazole prophylaxis has an antimalarial effect which could have an additional protective effect against malaria in HIV-infected children on antiretroviral therapy (ART). We measured the incidence and associated factors of malaria in HIV-infected children on ART and/or cotrimoxazole in Abidjan, Côte d’Ivoire.All HIV-infected children <16 years, followed-up in the IeDEA West-African paediatric cohort (pWADA) in Abidjan, were prospectively included from May to August 2012, the rainy season. Children presenting signs suggesting malaria had a thick blood smear and were classified as confirmed or probable malaria. We calculated incidence density rates (IR) per 100 child-years (CY). Risk factors were assessed using a Poisson regression model.Overall, 1117 children were included, of whom 89 % were ART-treated and 67 % received cotrimoxazole. Overall, there were 51 malaria events occurring in 48 children: 28 confirmed and 23 probable; 94 % were uncomplicated malaria. The overall IR of malaria (confirmed and probable) was 18.3/100 CY (95 % CI: 13.3-23.4), varying from 4.2/100 CY (95 % CI: 1.1-7.3) in children on ART and cotrimoxazole to 57.3/100 CY (95 % CI: 7.1-107.6) for those receiving no treatment at all. In univariate analysis, age <5 years was significantly associated with a 2-fold IR of malaria compared to age >10 years . Adjusted for severe immunodeficiency, cotrimoxazole reduced significantly the IR of first malarial episode . Severe immunodeficiency increased significantly the malaria IR .When considering the IR of confirmed malaria only, this varied from 2.4/100 CY (95 % CI: 0.0-4.8) in children on ART and cotrimoxazole to 34.4/100 CY (95 % CI: 0.0-73.3) for those receiving no treatment at all. In adjusted analyses, the IR of malaria in children on both cotrimoxazole and ART was significantly reduced  compared to those receiving no treatment at all.Cotrimoxazole prophylaxis was strongly protective against the incidence of malaria when associated with ART in HIV-infected children. Thus, these drugs should be provided as widely and durably as possible in all HIV-infected children <5 years of age. In 2010, approximately 216 million malaria episodes occurred worldwide, 81 % of which were in sub-Saharan Africa. Overall, 655,000 of these cases led to death, 86 % occurring in children under five years ), one in Cocody  and one in Abobo (the Mother To Child Transmission centre [MTCT-Plus]). In 2012, the incidence of malaria in Abidjan varied in the general population, ranging from 215 ‰ in Cocody area to 112 ‰ for Yopougon; in Treichville it was 118 ‰ and 185 ‰ in Abobo. Thus, Cocody and Abobo districts were a 2-fold higher endemic areas for malaria compared to other districts in Abidjan .Compared to the other sites, MTCT-Plus had the highest observed IR of 77.5/100 CY (95 % CI: 31.7-123.2) , in univariate analyses, compared to children receiving no treatment at all, the incidence of malaria was respectively reduced by 93 % among children receiving both cotrimoxazole prophylaxis and ART  and by 84 % in those on cotrimoxazole alone , .p < 0.01): the MTCT-Plus care centre where the children were also the youngest with a median age of two years, was significantly associated with a higher incidence of malaria .Age < 5 years was also significantly associated with a higher incidence of malaria  compared to children aged > 10 years. Incidence of malaria was also significantly associated with the follow-up clinic , and 95 % , respectively)  but this reduction was not statistically significant. The adjusted incidence of malaria was significantly highest in children with severe immunodeficiency for age when adjusted for ART and cotrimoxazole prophylaxis   Table . We obseTo our knowledge, this study is the first to specifically describe the association between the intake of cotrimoxazole prophylaxis and the incidence of malaria in HIV-infected children in several care programs in Abidjan. It has allowed documenting in a standardized manner any suspected malaria cases among HIV-infected children. We reported a high incidence rate of malaria (18.3/100 CY) that was strongly reduced in children on cotrimoxazole alone or combined with ART when adjusting for severe immunodeficiency. Incidence of malaria was twice as high in children under five years compared to those older than 10 years. The incidence of malaria was not reduced in children treated with ART only compared to those not on ART.Our study was conducted in a substantial sample size of more than 1000 HIV-infected children during the rainy season, a high transmission period of malaria.However, we also note study limitations. First, our estimation of the incidence of malaria is likely to have been underestimated for several reasons: due to logistical reasons, our study period did not cover the entire rainy season, which usually ends in November. We only documented all clinically driven symptoms but did not performed routine thick smears in asymptomatic children. We may also have missed malaria cases because they could have been treated outside the study centres or because of self-medication for malaria before seeking care, which is often practiced in Côte d’Ivoire, as reported in 15 % of the cases in our study. This could have led to false negative results of the thick blood smear performed also contributing to underestimation of the incidence of malaria. Second, in this study we used the gold standard for malaria diagnosis, which is microscopy. In a high endemic setting, where the population is immunocompromised, the use of additional laboratory tests such as rapid diagnostic testing or PCR could have allowed identifying additional cases of malaria, but this was not feasible for logistical reasons. Finally, there may also be a classification bias over-estimating the incidence of malaria: we defined probable malaria as any clinical event evoking malaria and solved by antimalarial, however 23 % were co-treated with antibiotics which could indicate that the event may have been another infection, solved by the antibiotics the child was co-treated with. Nevertheless, we conducted this study over a short period of time with a previous awareness campaign among the medical staff to improve and standardise all the diagnosis procedures of malaria. Consequently we conclude that our estimation is quite a reasonable minimal estimate. Unfortunately, also for logistical reasons, it was no feasible to compare these results with a group of HIV-uninfected children in the same community.et al. has also reported this previously in an adult population in Uganda [in vitro, have shown a significantly protective effect of protease inhibitor drugs [In our study, the global incidence of malaria (confirmed and probable) in HIV-1-infected children was 183 ‰ and 307 ‰ in children under five, respectively. This estimation seems to be higher than others reported in Cote d’Ivoire. The country is ranked the fifth among countries most affected by malaria compared to the rest of the world with an incidence estimated to be 116 ‰ in the general population in 2012, with a similar risk in children less than five: 288 ‰ –26. We an Uganda . If usedor drugs . This dior drugs .Severe immunodeficiency is also known as a risk factor for malaria , 33, 34.Children <5 years have a twice higher risk to develop malaria in our study and this is expected and highly consistent with what was reported by the WHO .Our results suggest that cotrimoxazole provides an ongoing beneficial protection against malaria after immune reconstitution in ART-treated children. This justifies its use for all children even if they are receiving ART, with a beneficial additive effect, as recommended by WHO since 2000 .Likewise, we reported the frequency of malaria occurrence in children already severely immunodeficient, suggesting that the use of ART must be widespread to all HIV-infected children with the aim to restore immunity as recommended by WHO in 2013 . In addiIn this study, we measured incidence density rates of malaria in HIV-infected children and analysed its associated factors. Cotrimoxazole prophylaxis strongly protected against malaria. We did not show a significant association between incidence of malaria and ART. But cotrimoxazole prophylaxis protection is more important if used in addition to ART. Also, immunodeficiency was an important risk factor of malaria incidence suggesting that the indirect effect of ART in restoring immunity justifies its use for all HIV-infected children. These two drugs should be provided as widely and as durably as possible in all children, as recommended by WHO . Likewis


Plasmodium lifecycle in the vertebrate host. But it is well known that the parasite’s enormous capacity of genetic variation in conjunction with the host genetics polymorphism is also associated with a wide spectrum of susceptibility degrees to complicated or severe forms of the disease. In this scenario, variations in genes of the major histocompatibility complex (MHC) associated with host resistance or susceptibility to malaria have been identified and used as markers in host–pathogen interaction studies, mainly those evaluating the impact on the immune response, acquisition of resistance, or increased susceptibility to infection or vulnerability to disease. However, due to the intense selective pressure, number of cases, and mortality rates, the majority of the reported associations reported concerned Plasmodium falciparum malaria. Studies on the MHC polymorphism and its association with Plasmodium vivax, which is the most widespread Plasmodium and the most prevalent species outside the African continent, are less frequent but equally important. Despite punctual contributions, there are accumulated evidences of human genetic control in P. vivax infection and disease. Herein, we review the current knowledge in the field of MHC and derived molecules  regarding P. vivax malaria. We discuss particularly the results of P. vivax studies on HLA class I and II polymorphisms in relation to host susceptibility, naturally acquired immune response against specific antigens and the implication of this knowledge to overcome the parasite immune evasion. Finally, the potential impact of such polymorphisms on the development of vaccine candidate antigens against P. vivax will be studied.The importance of host and parasite genetic factors in malaria resistance or susceptibility has been investigated since the middle of the last century. Nowadays, of all diseases that affect man, malaria still plays one of the highest levels of selective pressure on human genome. Susceptibility to malaria depends on exposure profile, epidemiological characteristics, and several components of the innate and adaptive immune system that influences the quality of the immune response generated during the  Plasmodium, malaria remains a major public health problem. Malaria transmission occurs in 96 countries and territories, and according to the latest estimates, 3.3 billion people are at risk of infection. Approximately 214 million cases and 438,000 deaths due to malaria occur worldwide, mainly of children under 5 years. The great majority of cases (88%) and deaths (90%) occurs in Africa, followed by Southeast Asia (10%) and Eastern Mediterranean region (2%)  genes. Therefore, in the present study, we review the current knowledge in the field of MHC molecules regarding P. vivax malaria.Considering the intense selective pressure, the number of cases and the mortality rates associated with ed death , 58. Thehrocytes . PopulatPlasmodium genus, P. vivax lifecycle is a complex process and requires an invertebrate and a vertebrate host for survival and perpetuation  system in humans, is an extremely polymorphic region encoding for the major molecules in charge of antigen presentation on the cell surface, and it has been one of the most intensively studied areas in the human genome , 70. LocThe polymorphism of HLA has been useful in the search of donors with compatible grafts in tracing population migration as well as in its potential relationship to pathogen-mediated selection. Further, assessing and comparing the polymorphism of HLA allows to better define the extent of the genetic variability in humans as well as the reasons of this diversity. The HLA region is associated with more diseases (mainly autoimmune and infectious diseases) than any other region of the genome –78.P. falciparum Liver Stage Antigen-1  by sequence variations in the central region of the gene  is the most abundant polypeptide present in the sporozoite covering. This protein is involved in the motility and invasion of the sporozoite during its entrance in the hepatocyte . The cspthe gene , 82. A sthe gene . More rethe gene   have been reported as abundantly expressed on the surface of merozoites and can contribute to the initial recognition of erythrocytes. MSP-1, MSP-3, and MSP-9 have been considered important vaccine candidates based on their location, on their recognition by antibodies from individuals naturally exposed to e-growth –95. There-growth . In conte-growth . Howevere-growth .P. vivax antigens. In a study performed by Ferreira and co-workers, no genetic restriction mediated by HLA-DRB1* and HLA-DQB1* against two constructions of P. vivax Reticulocyte Binding Protein-1 (PvRBP1) was verified in more than 500 HLA alleles from different individuals from communities in the Amazon region of Brazil  in the TNF promoter (–308G > A and –1031C > T) were associated with cytokine levels and temperature, but no association related to susceptibility were reported  when compared to P. vivax infected individuals , which is an RNA helicase encoded by the DDX39B gene, has been described as a negative regulator of inflammation by modulating expression of proinflammatory cytokines (such as TNF) . Therefosymptoms .Plasmodium infection. However, the absence of studies using only P. vivax infected/exposed individuals makes the definition of genetic polymorphism of HLA-class III genes associated specifically to this species particularly difficult. In P. falciparum studies conducted in Africa, a trend of association between LTA polymorphism with antimalarial IgG subclass levels was found but not confirmed by statistical tests  malaria in a population infected with P. falciparum or P. vivax was investigated. Although no differences between species were found, the results showed, for the first time, an association between alleles of CTL4 gene with malaria, which displayed a significant association with reduced risk for clinical malaria. In addition, two other associations with cytokines were identified, both within MHC class III region, that included TNF and the lymphotoxin alpha (LT-α/LTA) and beta (LT-β/LTB) genes, which are closely related  for allele binding score and population coverage. The most promising peptide sequences were included in a PvRBP-1 chimeric antigen containing the predicted promiscuous T-cell epitopes and known B-cell epitopes and presented no HLA restriction in naturally acquired immune response of exposed individuals (P. vivax antigens were selected based on proteome and transcriptome data of P. falciparum orthologs. After immunological confirmation, four peptides were experimentally confirmed as truly immunogenic peptides and were preselected for further preclinical trials (P. vivax and P. falciparum LDH sequences, T-cell epitope prediction indicated that 28 HLA alleles could recognize pLDH antigen epitopes. Interestingly, despite a large number of potentially common or similar epitopes, specific Pv-LDH and Pf-LDH epitopes were also predicted and, if experimentally confirmed, could be involved in future specific diagnostic rapid tests.Despite several T-cell epitopes from pre-erythrocytic –118, ase methods , 123–125epitopes . More reentified . Even wientified . If the entified . For exaentified . This coividuals . Moreovel trials . Lastly,l trials . Based oP. vivax.In summary, with the concomitant advent of whole-genome sequencing and advances in bioinformatics, the vaccinology field changed in the last few decades, providing the opportunity of describing novel antigens and improving the already known. Consequently, the focus in vaccine design shifted to explore antigens susceptible to antibody recognition and T-cell induction through comparative pan genome reverse vaccinology. Even though, in most of cases, experimental confirmation is necessary, high-accuracy predictions are available for any HLA known, non-human primates, mouse strains, and other mammals. Therefore, those “reverse immunology” systems have become highly accessible, and they can be a fast and efficient alternative when some conventional vaccinology strategies are difficult, especially when dealing with non-culturable microorganisms, as P. vivax specific immune response and clinical outcome are still increasing, and there are several similarities and disparities among these association studies. Despite the variation of MHC genes, alleles and/or haplotypes in different clinical and epidemiological scenarios, the association between MHC genes and P. vivax has been demonstrated in the majority of studies presented. We believe that the inconsistency of some data may derive from the fact that a large number of potential risk factors, such as nutritional status, coinfections, and relapses, which could influence the specific immune response, are almost impossible to be controlled in malaria endemic areas. Other issues are the small sample size, the heterogeneity of human populations in different endemic areas, and of course, the complexity of MHC genes. Therefore, since population-based cohorts with a single P. vivax infection represent a valuable but uncommon resource for genetic studies, more sophisticated analytical approaches are needed to study the expression of MHC genes in such different exposure conditions to determine the precise role of such polymorphisms as determinant for P. vivax susceptibility, immune response, and its outcome in disease progression.The number of studies involving MHC polymorphism and JL-J and LP-R wrote the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.