Plasmodium falciparum and Plasmodium vivax infections in south-central Oromia, Ethiopia.The presence of asymptomatic infections has serious implications for malaria elimination campaigns. Since asymptomatic carriers do not seek treatment for their infection and may become gametocyte carriers, they undoubtedly contribute to the persistence of malaria transmission in a population. The presence of asymptomatic parasitemias was noted in areas with seasonal malaria transmission. In Ethiopia there is a paucity of data regarding the prevalence of asymptomatic malaria carriage. This study was undertaken to assess the presence and prevalence of asymptomatic A total of 1094 apparently healthy individuals ≥ 2 years of age in south-central Oromia, Ethiopia, an area with seasonal and unstable malaria transmission, were screened for the presence of asymptomatic plasmodial infections. Finger-prick blood samples were taken from each participant for blood film preparation for microscopy and the rapid diagnostic test (RDT). Blood samples were also spotted on Whatman 3MM filter paper for parasite DNA extraction.Plasmodium carriage  was 5.0 %  as determined by microscopy, while the prevalence as determined using RDT was 8.2 % . PCR was done on 47 of 55 microscopy-confirmed and on 79 of 90 RDT-confirmed samples. PCR detected parasite DNA in 89.4 % (42/47) of the microscopy-positive samples and in 77.2 % (61/79) of the RDT-positive samples. No significant difference was observed in the prevalence of asymptomatic P. falciparum or P. vivax infections in the study area (P > 0.1). However, the prevalence of asymptomatic parasitaemia was significantly associated with gender  and age . Age and parasite densities had an inverse relationship.The prevalence of asymptomatic P. falciparum and P. vivax infections in south-central Oromia, an area with low, seasonal and unstable malaria transmission in Ethiopia. Of 55 microscopically confirmed asymptomatic infections, P. falciparum monoinfection accounted for 45.5 % and of 90 RDT positive asymptomatic infections, 66.7 % were P. falciparum. Although not statistically significant, P. falciparum accounted for a relatively large number of the asymptomatic infections as determined by both tests. The prevalence of asymptomatic parasitaemia was highest in the younger age group. HRP-2-based RDTs specific for P. falciparum showed high false positivity rate compared to Plasmodium lactate dehydrogenase (pLDH) specific to P. vivax. Although microscopy and RDT detected substantial numbers of asymptomatic infections in apparently healthy inhabitants, the use of a highly sensitive molecular diagnostics offers a more accurate assessment of the magnitude of asymptomatic infections.This study confirms the presence of asymptomatic  Plasmodium parasite antigens using one or more of three target antigens: histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH), and aldolase. HRP2 is expressed only by Plasmodium falciparum and is the most widely used target antigen for malaria RDTs. LDH and aldolase are expressed across all Plasmodium species but tend to yield lower diagnostic accuracy in commercially available RDTs.Substantial reductions in malaria transmission and morbidity have been reported in many parts of East Africa, consequent to the spirited up-scaling of interventions in the region –4. DespiAlthough patients with subclinical infections do not present with malaria symptoms, they still contribute to the cycle of transmission in a population. The relative contribution of sub-clinical infections has considerable implications for the design and use of elimination diagnostics. The epidemiology of asymptomatic malaria in different transmission settings is attracting increasing attention, because asymptomatic individuals are still able to produce gametocytes and therefore provide the reservoir for onward transmission –14. The Plasmodium per se would end up with clinical malaria. The objective of this study was to determine the prevalence of asymptomatic Plasmodium spp. infection using microscopy and RDT in a population-based cross-sectional survey in south-central Oromia, Ethiopia. PCR was not conducted on all microscopy and RDT positive samples but a subset of these. The fact that some of the microscopy and RDT positive individuals were anaemic we were unable collects blood spots for molecular methods and hence not included in the PCR analysis. It is anticipated that data from this study will have important practical implications to the malaria elimination strategies in Ethiopia.Given the importance of asymptomatic malaria infection in different endemic areas of the world, various studies have assessed the prevalence of asymptomatic infections in the control and elimination phase of malaria, because detection and treatment of all sources of infection is very critical at this stage , 19–22. kebeles  of the Shalla District from November through December 2012. The kebeles have known population sizes and systematically registered households. Each kebele is sub-divided into villages. After obtaining informed consent from parents/guardians, all members of the randomly selected households were requested to give finger-prick blood samples. Inclusion criteria were that all volunteers must (1) be apparently healthy , and (2) residing in the village for more than one year, and (3) consent to giving a blood sample. Members of a household who had contracted malaria and were treated within the previous month and/or were unwilling to participate in the study were excluded. Individuals with body temperature ≥37.5 °C as measured by digital thermometer were also excluded.This study was conducted in West Arsi Zone, Oromia Region, Ethiopia, located approximately at a distance of 251 km from the capital city, Addis Ababa. Malaria transmission is seasonal and unstable in this area. A cross-sectional study was conducted in 12 The study area has unique feature, there is a severe shortage of surface water bodies. To overcome shortage of water bodies in the surrounding, inhabitants dig ground and use it as a reservoir to store rain water. This man-made reservoir ground dries out after a few months of the termination of the rain seasons Fig. .Fig. 1MaPlasmodium spp. infections. Using 95 % confidence level, a design effect of 1.5, a margin of error of 4 %, and anticipated asymptomatic prevalence of 50 %, a total of 993 individuals were screened (using the formula N = (Zα/2)2P (1-P)*DEFF/ME2 where N is the sample size, Zα/2 is the critical α level, P is the anticipated asymptomatic malaria prevalence, DEFF is the design effect and ME is the marginal error). But to include 993 individuals in the sample, it was proposed that 1094 individuals were invited to participate, thus allowing for a 10 % non-response. Finger-prick blood samples were used for microscopy and RDT tests. For PCR analysis, blood samples were spotted onto Whatman 3MM filter paper. The filter papers were dried and stored individually in sealed plastic bags. Examination of the blood films was performed by experienced malaria microscopists. Participants tested malaria positive by RDT were treated according to the national treatment guideline: Artemether-lumefantrine for P. falciparum and chloroquine for P. vivax infected patients.A community-based cross-sectional study was conducted to determine the prevalence of asymptomatic Thick and thin blood smears were made on the same slide, air dried and transported to the Adama Malaria Control Center. The slides were then stained with 10 % Giemsa for 15 minutes and screened for the presence of plasmodial infections. Two experienced microscopists read the slides blinded to individual’s RDT results and to each other. Fortunately, the agreement of the two microscopists was 100 % in species identification. Parasite density was determined from thick blood smears by counting the number of asexual parasites per 200 WBCs and calculated assuming a standard mean white blood cell count of 8,000 leukocytes per μl of blood . A smearP. falciparum and Plasmodium lactate dehydrogenase (pLDH) specific for P. vivax.The SD BIOLINE Malaria Ag P.f/P.v POCT test kit  was used to capture malaria antigens and was performed in accordance with the manufacturer’s instructions. The kit targeted malaria antigens HRP-2 specific for Plasmodium species were identified by microscopy and species-specific nested-PCR [2 piece of filter paper with blood spot was cut with an automatic cutter and placed in 1 ml of phosphate buffered saline containing 0.5 % saponin and incubated overnight at 4 °C. The brown solution was removed and replaced with 1 x PBS and then incubated for 1 h at 4 °C. The solution was removed and 150 μl of DNAse free water was added followed by 50 μl of 20 % Chelex. The tubes were placed into a heated block (99 °C) for 10 mins and vortexed every two min. This was repeated 2–3 times. The solution was centrifuged at 13, 000 rpm for 10 min. The supernatant was carefully separated and centrifuged as before. The supernatant which contains DNA was collected carefully into 0.5 ml clean tube and used for PCR immediately or stored at −20 °C until use. The extracted DNA was amplified by nested-PCR . Primer sequences (5’-3’) used for the primary PCR in P. falciparum (outer): CCGTTAATAATAAATACACGCAG and CGGATGTTACAAAACTATAGTTACC (reverse). For the nested PCR (outer): TGTGCTCATGTGTTTAAACTT and CAAAACTATAGTTACCAATTTTG (reverse) primer sequences were used. For P. vivax, primers for primary PCR (outer): CGCCATTATAGCCCTGAGCA and TCTCACGTCGATGAGGGACT (reverse). For the secondary PCR (outer): GGATAGTCATGCCCCAGGATTG and CATCAACTTCCCGGCGTAGC (reverse). Primary and nested PCR assays were performed in a 20 μl volume reaction mixture of DreamTaq buffer, 100 μM dNTPs, 0.05 μM of each primers and 1.25 units of Dream-Taq enzyme (Thermo Scientific). Two microliters (2 μl) of the sample DNA were used for outer amplification. DNA from P. falciparum strain 3D7 and sterile water were used as positive and negative controls, respectively. For P. vivax, microscopy and RDT known samples were used as positive control. Amplicons were resolved in ethidium bromide-stained 1.5 % agarose gel and observed under ultraviolet transillumination.Parasite DNA was extracted using the Chelex method . PCR wassted-PCR . BrieflyThis protocol was reviewed and approved by the Institutional Review Boards (IRBs) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University and of the Armauer Hansen Research Institute, as well as by the National Research Ethics Review Committee (NRERC). Information about the objective of the study was given to the head of each household. Written informed consent was obtained from all adults willing to participate in the study, with a parent/guardian giving consent for children. Malaria positive subjects were treated as per the national treatment guideline.K), assuming microscopy as a ‘gold standard’. Bivariate and multivariate logistic regressions were performed to estimate the association between the presence of asymptomatic Plasmodium carriages and variables of interest. An error probability  of <0.05 was considered as statistically significant.Data were checked for completeness and consistency, and double entered into a SPSS17.0  database. Descriptive statistical analysis was done using STATA 11.0 . Fisher’s exact test was used to assess the difference in the prevalence of asymptomatic infections between variable of interest. The agreements between microscopy and RDT data were assessed using Cohen’s kappa coefficient  with microscopically and/or RDT confirmed Plasmodium infection. By microscopy, the prevalence of asymptomatic parasitaemia  was 5.0 % (55/1094). Out of a total of 55 microscopy-positive samples, 45.5 % (25/55) were P. falciparum, 40 % (22/55) were P. vivax and 14.5 % (8/55) mixed  infections . Children below 5 years had the highest (12.7 %) whereas over 35 year-old adults had the lowest infection rates (2.4 %) as determined by microscopy.A total of 1, 094 individuals  from 317 households were surveyed in 12 randomly selected villages/P. falciparum, P. vivax and mixed infections, respectively. RDT was positive in 85.5 % (47/55) of microscopy-positive samples. Microscopy and RDT had 95.2 % agreement (Kappa = 0.6248). Considering both microscopy and RDT data, no significant difference was observed in the prevalence of asymptomatic P. falciparum or P. vivax infections in the study area (Fisher’s 2-tailed P = > 0.1).Using RDT, the prevalence was 8.2 %  and this was significantly higher than the infection prevalence  detected by microscopy . Out of 90 RDT-confirmed positive subjects, 66.7 %, 25.6 % and 7.7 % were Plasmodium spp. infections, PCR was done on 47 and 79 of the samples, respectively. PCR detected parasites DNA in 89.4 % (42/47) of the microscopy-positive samples. On the other hand, PCR detected parasites DNA in 77.2 % (61/79) of the RDT-positive samples. Thus, the true positivity rate for microscopy and RDT relative to PCR were 89.4 % and 77.2 %, respectively . For parasitaemia between 500–1000 parasites per μl of blood, two were P. falciparum and three were mixed infection. From a total of 13 subjects with parasitaemia between 1000–5000 parasites per μl of blood, 6 P. falciparum and seven were P. vivax infections. Of 17 individuals with parasite density greater than 5000 parasites per μl of blood, four P. falciparum, nine P. vivax and four were mixed infections. The distribution of parasite densities with respect to age groups and sex is shown in Fig. Age and parasite densities had an inverse relationship. Of 11 subjects with parasitaemia between 100–500 parasites per μl of blood, five P. falciparum, 22 were P. vivax and 8 were mixed infections. Of 17 asymptomatic P. falciparum cases, 58.8 % (10/17), 23.5 % (4/17) and 17.6 % (3/17) had asexual parasites, gametocytes and both asexual and gametocytes, respectively. From a total of 22 asymptomatic P. vivax cases, 86.4 % (19/22) had asexual parasites and 13.6 % (3/22) had both asexual and gametocytes. Six of eight mixed infections (75 %) had both asexual and gametocytes and 25 % had asexual parasites alone. The mean density of P. falciparum was 8074 parasites per μl of blood and 4821 parasites per μl of blood for P. vivax (data not shown).From a total of 47 subjects whose parasitaemias were determined, 17 were P. vivax was identified. In W/bute, Chefa and O/shibibo villages, only P. falciparum was identified. On the other hand, both P. falciparum and P. vivax were found in A/rima C/kuntufa S/kamala, Walilalti and M/binsho villages. Fig. P. falciparum and P. vivax were noted in some of the study villages. There were many instances of detecting more than one P. falciparum or P. vivax infection within a given household. The prevalence of asymptomatic infections ranged from as low as 0 % (D/bunge) to as high as 8.5 % (A/rima) as determined by microscopy.Asymptomatic parasitaemia was identified in 19.9 % 63/317) of the households surveyed. In W/kosha and Machafara villages only /317 of tP. falciparum and P. vivax infections in the West Arsi Zone of the Oromia Region of Ethiopia, an area where malaria transmission is markedly seasonal and unstable. In this study, there was a slight predominance, although not statistically significant, of falciparum over vivax infections as diagnosed by both microscopy and RDT unlike in Brazil where P. falciparum accounted for large number of the asymptomatic infections as diagnosed by microscopy [P. falciparum and P. vivax infections determined in this study is in agreement with a study in India that showed low prevalence (4.3 %) of asymptomatic P. falciparum and P. vivax infections [Given the renewed focus on transmission reduction leading to elimination, the quantification of the malaria parasite reservoir in a given area is important for control. In areas where malaria is sustained at low levels or is highly seasonal, asymptomatic reservoirs of infection are critical for maintaining malaria transmission . Since scroscopy , 27. Thefections .Most children in areas with moderate-to-high levels of malaria transmission gain protection from severe disease usually by 2–5 years of age, followed by a decrease in the rate of symptomatic illness in early adolescence . Given tFalse positivity values were 10.6 % (5/47) for microscopy and 22.9 % (18/79) for RDT relative to PCR. Despite its many advantages, the limitations and shortcomings of microscopy are well documented . The perP. falciparum, six P. vivax and one mixed infection in 47 of microscopy negative samples (that were positive by RDT), suggesting that asymptomatic parasitaemia in these samples could be below the threshold of microscopy. PCR confirmed the presence of parasite DNA in 89.4 % of microscopy-positive samples. Considering the fact that PCR can detect parasitemias as low as 0.02 parasites per μl, it is not surprising to note the superiority of the PCR over the other classical diagnostic methods [P. falciparum, four P. vivax and one mixed infections in 15 of the RDT-negative samples, suggesting that parasite antigens in these samples could be below the detection limit of RDT. Moreover, PCR failed to detect two of the microscopy positive P. falciparum samples and three of the microscopy positive P. vivax samples. The lack of detection of infections by PCR in microscopy and RDT positive samples could be due to the small amount of DNA used in the PCR reaction as evidenced by Hodgson et al. [In this study, PCR detected 20  methods . PCR detn et al.  where hiP. falciparum samples were found to have either P. vivax or mixed infections. Four of 19 (21 %) microscopy-positive P. vivax samples were found to have either P. falciparum or mixed infections by PCR  microscopy-positive CR Table . Consequ disease , 44. A mortality . A recenortality  showed tortality –50.P. falciparum and P. vivax infections. Of particular interest regarding the transmission potential, 41.2 % of the P. falciparum and 13.6 % of the P. vivax infections had gametocytes detected either alone or along with the asexual stages of the parasite. Moreover, gametocytes were detected in 75 % of mixed infections (Pf-Pv). The presence of asymptomatic cases with circulating sexual stages of the parasite (gametocytes) shows their potential to infect mosquitoes and cause additional human cases.In line with the importance of asymptomatic infections to the infectious reservoir, the presence of gametocytes was analyzed for both P. falciparum and P. vivax infections were either colocalised or unique to certain study villages. In W/kosha and Machafara villages only P. vivax was identified. In W/bute, Chefa and O/shibibo villages, only P. falciparum was identified. On the other hand, both P. falciparum and P. vivax were found in A/rima C/kuntufa S/kamala, Walilalti and M/binsho villages. As to why P. falciparum and P. vivax co-exist or are unique to certain study villages needs further study. However, factors  like variations in water bodies  around the study villages may have accounted for clustering of asymptomatic infections. Indeed, geographic clustering of symptomatic and asymptomatic malaria has been reported elsewhere [It is apparent that distance  to water is a major determinant of malaria risk. In this study, the presence of asymptomatic parasitaemia shows clustering around a few study villages. Moreover, lsewhere , 51. In Overall, the 5 % prevalence of asymptomatic parasitaemia as determined by microscopy in this study was substantially lower than that reported from other African countries. The prevalence of asymptomatic parasitaemia as determined by microscopy was 12 % in Gabon , 12.6 % P. falciparum and P. vivax infections do exist in the study area. As determined by microscopy, P. falciparum accounted for 45.5 % of asymptomatic infections, P. vivax 40 % and mixed infections 14.5 %. Asymptomatic parasitaemias were high at younger age groups, and men are at a greater risk of having asymptomatic Plasmodium spp. infections than women. The transmission of Plasmodium spp. from humans to mosquitoes requires the presence of infectious gametocytes in the human peripheral blood. Thus, the detection of asymptomatic cases with circulating gametocytes signals their importance in maintaining sustained transmission in the study area. Indeed, the presence of asymptomatic cases is a big challenge for the management of elimination programs in any malaria endemic area. As a result of a missed diagnosis by RDT on field condition, some P. vivax patients were treated as P. falciparum and vice versa as evidenced by PCR. As malaria control efforts are gaining momentum and elimination agenda is initiated, further research is needed to better understand the epidemiology of asymptomatic reservoir in Ethiopia. Although microscopy and RDT detected considerable numbers of asymptomatic infections in apparently healthy individuals, the use of a highly sensitive molecular diagnostics in large epidemiology studies offers a more accurate assessment of the magnitude of asymptomatic infections. Further studies are needed for a better understanding of the asymptomatic Plasmodium spp. infections and their contribution to the dynamics of malaria transmission and to the incidence of symptomatic infections.The main conclusions of this study were that asymptomatic


Plasmodium vivax, considered a harder-to-eliminate parasite. This study aim is to describe the transmission dynamics and associated risk factors in a rural settlement area in the Western Brazilian Amazon.In recent years, considerable success in reducing its incidence has been achieved in Brazil, leading to a relative increase in the proportion of cases caused by P. vivax and Plasmodium falciparum malaria within 90 days of a previous episode were analysed using univariable and multivariable Cox-Proportional Hazard models.A prospective cohort was established in a rural settlement area for 3 years. Follow-up included continuous passive case detection and monthly active case detection for a period of 6 months. Demographic, clinical and transmission control practices data were collected. Malaria diagnosis was performed through thick blood smear. Univariable and multivariable analyses of factors associated with malaria incidence were performed using negative binomial regression models. Factors associated with recurrence of P. vivax predominating and P. falciparum disappearing after 1 year of follow-up. Malaria incidence was significantly higher in the dry season . Use of ITN was associated to malaria protection in the localities . A recurrent P. vivax episode within 90 days was observed in 29.4 % of individuals after an initial diagnosis. A previous P. vivax  or mixed P. vivax + P. falciparum  infections were significantly associated to a vivax malaria episode within 90 days of follow-up.Malaria prevalence decreased from 7 % at the study beginning to 0.6 % at month 24, with P. falciparum and P. vivax co-endemicity, a virtual disappearance of P. falciparum was observed with P. vivax increasing its relative contribution, with a large proportion of recurring episodes. This finding reinforces the perception of P. falciparum being more responsive to early diagnosis and treatment and ITN use and the contribution of relapsing P. vivax to maintain this species’ transmission. In areas of P. vivax endemicity, antihypnozoite treatment effectiveness assessment in different transmission intensity may be a fundamental activity for malaria control and elimination.In an area of The online version of this article (doi:10.1186/s12936-016-1326-2) contains supplementary material, which is available to authorized users. Malaria is a preventable, diagnosable and treatable disease. With innovation and roll out of interventions there are fewer people dying from malaria now than in any other historical period. During 2015, there were an estimated 214 million cases of malaria and an estimated 438,000 deaths  globally  in comparison with P. vivax malaria .The risk of malaria infection was significantly higher in the dry season  and wherever IRS had been used in the last 6 months . Use of ITN was associated to malaria protection in the localities . Gender, age and socioeconomic status were not related to malaria incidence. Stratification by malaria species showed similar results Tables . HoweverP. vivax within 90 days of a previous episode was 189/642 (29.44 %)  Fig. . RecurreP. vivax  or mixed P. vivax + P. falciparum  infections were significantly associated to a vivax malaria episode within 90 days of follow-up. Recurrent P. vivax infections were also significantly higher in the dry season   2.5 1.–3.5; p =P. falciparum and P. vivax, and in a context of a marked decrease incidence between the beginning and the end of study activities. This confirms P. vivax as a more resilient species, with the virtual disappearance of P. falciparum, a reducing proportion of malaria-attributed fever and an increasing clustering of malaria episodes with reduced incidence. While P. falciparum and P. vivax responded each for around 50 % of cases in Brazil in 1988 this situation started changing in the 1990s, leading to the current situation in which P. vivax has become the predominant species in recent years  [This study was able to comprehensively characterize the epidemiology of malaria in an area coendemic for ciparum) , 5, 17. P. falciparum (~50 %) when comparing to P. vivax (~30 %). Plasmodium vivax biological features may have contributed to lower vector control effectiveness in this group. Additionally, among anopheline collections in these communities, there was a predominance of mosquitoes in the peri-domiciliary environment [In Brazil, current methods for vector control are based primarily on the use of long-lasting insecticide-treated nets (ITN) and indoor residual spraying (IRS), but there is no specific and systematic evaluation on the impact that these measures have on suppressing anopheline populations and reducing levels of malaria parasite transmission in recent agrarian reform projects in the Amazon . In thisironment .P. falciparum transmission, as gametocytes for this species are usually not detected at patent levels before 5 days of the onset of symptoms, as compared to P. vivax, for which it occurs much earlier [Opportune diagnosis and treatment, especially if provided within 48 h of symptoms onset, is thought to have a relatively higher impact on  earlier , 19. In  earlier . UnfortuP. vivax that contributes to this species burden and less notable responsiveness to the usual control measures [P. vivax with reducing number of cases, which in some months accounted for up to 50 % of episodes, irrespective of bed net use and routine primaquine prescription. It has been shown through elegant investigations that relapses may account for up to 80 % of vivax malaria episodes in Papua New Guinea [P. vivax episode (mono or mixed-infection) implied a considerable higher risk of presenting a new P. vivax clinical episode. Interestingly, for the specific analyses of recurrence, bed net use was not associated with protection, what would be consistent with the suggestion of a significant proportion being caused by relapses.Besides the later gametocytemia, hypnozoites responsible for the occurrence of relapses are also believed to be an important specific feature of measures . This isw Guinea , where pw Guinea , 22. Ourw Guinea . Our anaP. vivax infection after a monoinfection due to P. falciparum, in agreement with previous observations from Thailand [P. falciparum infection, especially in high density, is a proxy marker of malaria naivety and hence poor immunity to both P. falciparum and P. vivax infections. If this is true, relapses due to P. vivax hypnozoites acquired at or around the same time as the index P. falciparum infection would have a greater chance of reaching patency [P. vivax hypnozoites [Interestingly, individuals were significantly more likely to have recurrent Thailand . The patThailand . One pot patency . Historinozoites .P. vivax malaria relapses [In non-endemic areas in Brazil, inadequate primaquine dosing was found to be associated with relapses , 9. Unforelapses , as wellrelapses , factorsPlasmodium infection. In a different rural Amazon location, researchers have reported that conventional microscopy missed 54 % of Plasmodium carriers, indicating a large proportion of low-density infections in low transmission areas [Plasmodium in the area is likely to be considerably higher than the reported here. Positive microscopy in asymptomatic Plasmodium carriers was observed previously in the Brazilian Amazon [In the cross-sectional evaluations, a low frequency of fever  was observed among individuals with patent on areas . These fn Amazon –33.P. falciparum and an increase of the incidence of P. vivax (Additional file One important finding of this study is that the reducing incidence of malaria was accompanied by a dramatic decrease on the overall incidence of acute fever episodes of 80 % from the first to the third year of follow-up. During the same period, the proportion of malaria-attributed fever also decreased considerably, from around 70 % during the high season in the first year of study to no higher than 20 % in the third year of follow-up. These findings have important implication for the local health system and transmission control activities. This figure surely reflects a high proportion of non-diagnosed fevers, and the ability of health systems to identify the causes of fever is important for both adequate individual case management and for health surveillance, as demonstrated in other areas –38. ThisP. vivax is more challenging than tackling P. falciparum, and may require specific tools and strategies for its containment, especially regarding the prevention of relapses [P. vivax phenotypes may be more widespread and more prevalent in the Amazon than currently thought. This is an issue that remains to be more adequately investigated using enhanced approaches that would comprise pragmatic intervention trials and molecular methods [P. falciparum in late 2015. Whether focusing on this species is a more appropriate strategy than attempting to eliminate overall malaria is yet to be determined. In order to achieve a rapid and sustained elimination of malaria transmission in the region, health systems and control strategies will be required to adapt to changing epidemiological scenarios through integrated and innovative approaches for which design scientists, health authorities and communities will have to be working together towards its achievement.Data presented here represent an important contribution to the knowledge of the epidemiology of malaria transmission and vector control in agricultural settlements in the Amazon and could be useful to assist on the development and implementation of strategies to control and eliminate this infection in the region. Eliminating relapses , 42. A t methods , still t


Plasmodium ovale is one of the causative agents of human malaria. Plasmodium ovale infection has long been thought to be non-fatal. Due to its lower morbidity, P. ovale receives little attention in malaria research.Plasmodium ovale parasites were identified from blood smears of these patients. The species identification was further confirmed with nested PCR. One of them was successfully treated with no incident of relapse within 12-month medical follow-up. The other patient came down with malaria-induced respiratory complication during the course of treatment. Although parasites were cleared off the circulation, the patient’s condition worsened. He succumbed to multiple complications including acute respiratory distress syndrome and acute renal failure.Two Malaysians went to Nigeria for two weeks. After returning to Malaysia, they fell sick and were admitted to different hospitals. P. ovale curtisi.Sequencing of the malaria parasite DNA from both cases, followed by multiple sequence alignment and phylogenetic tree construction suggested that the causative agent for both malaria cases was P. ovale in causing severe complications and death as seen in this case report was highlighted.In this report, the differences between both cases were discussed, and the potential capability of Plasmodium ovale is potentially capable of causing severe complications, if not death. Complete travel and clinical history of malaria patient are vital for successful diagnoses and treatment. Monitoring of respiratory and renal function of malaria patients, regardless of the species of malaria parasites involved is crucial during the course of hospital admission. Plasmodium falciparum infection. This potentially grave complication has also been reported in malaria caused by Plasmodium vivax, Plasmodium malariae, Plasmodium knowlesi and Plasmodium ovale[P. ovale has been overshadowed by other human malaria parasites in the field of medicine and medical research. Nevertheless, recent studies have shown that ovale malaria is caused by two genetically distinct subspecies, P. ovale curtisi and P. ovale wallikeri[Acute respiratory distress syndrome (ARDS) is one of the severe complications of malaria. ARDS inium ovale-12. Due ium ovale, as wellium ovale, P. ovalwallikeri-18.P. ovale infection acquired from the same location were presented. Both cases ended with different outcome, and two interesting turning points were ARDS complication and acute renal failure.In this report, two cases of Two Malaysian acquaintances (patients A and B) went to Victoria Island, Nigeria together for a two-week working trip. Lariam® (mefloquine) was used as anti-malarial prophylaxis for the trip. They fell sick after returning to Malaysia and were admitted to different hospitals. Their cases are presented as follows:About two months after the trip to Nigeria, patient A  was admitted to a hospital due to five consecutive days of fever with chills and rigours. He was jaundiced, anorexic and febrile with body temperature of 37.7°C upon admission. He had mild cough, blood pressure of 110/66 mm Hg, pulse rate of 98 beats per minute (BPM) with peak bilirubin level of 45 μmol/L and hepatosplenomegaly. His lung examination was normal. His urine was tea-coloured. Ultrasound study confirmed the findings of hepatosplenomegaly with signs of chronic cholecystitis and cholelithiasis. Initial haematological investigation showed that he was thrombocytopaenic  with normal white blood cell (WBC) count  and haemoglobin level of 13.9 g/dL. He had not travelled to any other places after the trip to Nigeria. The patient had a past history of malaria for three times. The last episode of malaria was six months prior to present admission. However, the species of malaria parasites for the previous malaria episodes was not known. The patient also had an underlying condition of hypertension. Besides, he was a heavy alcohol consumer. Clinical findings on patient A upon admission are summarized in Table Plasmodium vivax-like” parasites were found with parasitaemia of 0.10%. Further microscopic examination by a referral diagnostic centre subsequently indicated that this was a mono-infection of P. ovale. This was confirmed with nested PCR technique using primers developed from the 18S ribosomal RNA (18S rRNA) gene as applied by previous reports[Patient A was treated immediately for cholecystitis with intravenous (IV) ceftriaxone 2 g daily and IV metronidazole 500 mg thrice daily by the attending gastroenterologist. However, his fever and thrombocytopaenia persisted, and WBC count dropped progressively. On day 5 of admission, blood smears were prepared and examined under the microscope. “ reports-21, coup reports. MeanwhiHe was treated with a course of six doses of Riamet® (artemether and lumefantrine), four tablets per dose, and primaquine for two weeks. Patient A responded well to the anti-malarial treatment clinically and biochemically. Patient’s parasitaemia dropped to 0.06% the following day. Malaria parasites were cleared in less than 48 hours after initiation of Riamet® treatment. He was discharged well on day 8 of hospitalization. He remained well without relapse of malaria throughout his medical follow-up of 12 months with the hospital.P. ovale, which was further ascertained with nested PCR as mentioned in the previous section. Clinical findings on patient B upon admission are summarized in Table Around six months after the trip to Nigeria, patient B  fell sick and went to a private hospital. He was then referred to a tertiary referral hospital. Upon admission to the referral hospital, he gave a history of intermittent fever with rigours, myalgia and nausea for ten days. His blood pressure upon admission was 102/55 mm Hg, with pulse rate of 60 BPM. Plasma glucose level was 9.5 mmol/L. Jaundice and hepatosplenomegaly were not detected. He was alert and conscious. His lung examination was normal. He made a one-day-trip to Kota Kinabalu, Sabah, three months before the admission. He had no known medical illness and no known history of acquiring malaria. Initial haematological investigation revealed that he was thrombocytopaenic  with low WBC count  and haemoglobin level of 12.4 g/dL. Malaria parasites were detected in his blood, with parasitaemia of 0.18%. The species was identified as Anti-malaria therapy course of chloroquine (chloroquine phosphate 150 mg base) and primaquine (30 mg) was started on patient B. However, he was still febrile (38.4°C) 24 hours later. On day 3 of the admission, patient B developed loose stools and lung examination revealed fine basal crepitations. Blood smear examination showed that the malaria parasites were not cleared. In the morning of day 4, he complained of feeling breathless and lethargic. His body temperature surged to 39.2°C. With presence of basal crepitations, an initial diagnosis of pneumonia was made and IV ceftriaxone was added into the course of treatment.However, later that day in the afternoon, the patient developed worsening dyspnea, haemoptysis, and subsequently epistaxis. Chest X-ray examination showed bilateral haziness up to the upper zone, which was suggestive of pulmonary haemorrhage. Haematological investigation showed that his platelet count was 120,000/μl and the malaria parasite load was reduced to 0.03%. Despite the lowering of parasitaemia, patient B progressed into respiratory failure. He was intubated and ventilated. His anti-malarial treatment was changed to IV quinine 850 mg (1 dose) and subsequently to IV artesunate 160 mg (for 7 days). Furosemide was given to the patient for presumed pulmonary oedema.On day 5 of the admission, malaria parasites were completely cleared. However, patient was still febrile with temperature of 40.8°C. He was transferred to intensive care unit. On day 6, patient B was oliguric. Increased level of creatinine and worsening of respiratory acidosis were noted. The patient had developed acute kidney injury (AKI) secondary to overwhelming sepsis. Hypotensive episodes were encountered. Therefore inotropic support was initiated. His antibiotic regime was changed to IV Tacocin® (tazobactam and piperacilin).On day 12, patient B was still febrile with little improvement on his lung function. He was still dependent on the ventilator. IV Tacocin® was replaced by IV vancomycin and imipenem empirical treatment. All the five sets of bacteriological blood cultures and one set of fungal culture requested earlier on different days came back as negative. Nevertheless, dialysis was started on day 15 due to oliguric AKI with worsening acidosis (mixed respiratory and metabolic). Dialysis was performed on daily basis till the end of his life.Enterobacter cloacae, which was sensitive to carbapenems. Hence, the imipenem therapy was continued. However, due to epileptic seizures suffered by the patient on day 17, the antibiotic regime was subsequently replaced with meropenem. A computerized tomography (CT) scanning on the patient’s brain showed no abnormality. Another thorax CT scanning showed extensive bilateral lung consolidation and loculated pleural effusion. His fever persisted, and he was on prolonged ventilation with difficulty in weaning off. His condition continued to deteriorate. He went into recalcitrant atrial fibrillation on day 22 and required tripleinotropic support. Further blood bacteriological and fungal cultures were all negative. On the 23rd day of hospital admission, he went into asystole and succumbed to P. ovale infection with ARDS, acute renal failure, metabolic acidosis, and nosocomial sepsis.On day 17, bacteriological culture from patient’s blood sample that was collected on day 15 was positive for Consent was granted by patient/ patient’s family for the publication of these case reports.Plasmodium ovale is not found locally in Malaysia. Alignment of short segments from 18S rRNA gene sequences of these two cases, i.e. isolate MAL-1 (GenBank accession number KF192072) and isolate MAL-2 (GenBank accession number KF192073) with 18S rRNA gene sequences of other isolates of P. ovale spp. that were available in GenBank  were conducted. The multiple sequence alignment showed that the aetiological agents of the two cases in this case report were P. ovale curtisi  as described previously[eviously  who showed no complications[P. ovale infection is the ability of P. ovale to form hypnozoites, which can survive chemoprophylaxis[A case of ovale malaria imported from Nigeria was previously reported in Malaysia, and theications, both caphylaxis.P. ovale spp. can form hypnozoites and result in long latency[In case A, there was a delay in malaria diagnosis. This case is a good example of clinical setting where malaria tends to be overlooked at the initial stage of medical investigation due to low level of suspicion. From the initial haematological investigation, all but one (platelet count) showed values within the normal range. In many hospital settings, especially those of urban areas and non-malaria endemic regions, such investigation outcome usually carries a thin possibility of instigating a blood film microscopic examination. Indeed, a case of traveller’s malaria that developed into severe complications due to delayed diagnosis was reported in Croatia recently. The ava latency,26, whicPatient B succumbed to the malaria infection and he did not have any medical problems prior to the illness. Patient A, who is a heavy drinker, was known to have hypertension even before the infection, survived. One possible explanation for this is the partial immunity gained by patient A from previous incidents of malaria. People who have past history of acquiring falciparum malaria and those staying in malaria-endemic regions are known to show less severe symptoms during subsequent malaria infections compared to people who are from non-malaria endemic regions . Indeed,One important note worth mentioning here is the trend of ARDS onset and development in this case. Similar to the situation of malaria-induced ARDS reported previously,31,32, pEnterobacter cloacae infection was diagnosed during the course of his hospital stay. Here, bacterial infection was ruled out as the primary cause of ARDS in patient B as the bacteriological culture showed positive results as late as day 15, whereas he showed symptoms of acute lung injury as early as day 4. Undoubtedly, the bacterial infection that occurred at such critical timing would worsen the health condition of the patient, which acted as the secondary contributing factor towards the fatal end for this case. Another point worth mentioning was the onset of the ARDS symptoms after the initiation of anti-malarial therapy, which was similar to many cases of malaria-associated ARDS reported previously[In case B, nosocomial eviously,10,30-34eviously. In addiP. falciparum, P. vivax, and P. knowlesi[P. falciparum and to a lesser, but significant extent, P. vivax[Thrombocytopaenia was seen in both cases. Such phenomenon has been observed in patients infected with  knowlesi,21,36-39 knowlesi, rosetti knowlesi-44, and  P. vivax. This ev P. vivax,30.P. ovale infection that deserves attention is the acute renal failure. Acute renal failure is another potentially fatal complication in malaria[P. vivax and P. malariae infections[P. ovale infection with acute renal injury. In case B, the patient suffered renal injury a few days before he came down with nosocomial sepsis. Therefore, the acute renal injury seen in this case was less likely to be caused by nosocomial sepsis. Nevertheless, the nosocomial sepsis might act as the secondary contributing factor towards the renal failure in this patient. Besides, the medications used on the patient such as vancomycin is potentially nephrotoxic[Another aspect of this fatal case of  malaria,46,47. H malaria, the new malaria,50, and fections,52. A fafections. This ishrotoxic, thus seP. vivax, P. malariae and P. ovale are relatively insusceptible to chloroquine[Apart from the differences in pathological development of both cases, the anti-malaria treatment regimes used in both cases were dissimilar as well. For patient A, Riamet® with primaquine was used whereas for patient B, chloroquine and primaquine were prescribed at the initial stage of treatment. Parasites were not detected from patient A’s blood smear in less than 48 hours after initiation of antimalarial treatment. On the other hand, complete parasite clearance could only be declared on patient B after switching the medication to quinine and artesunate. Consequently, the parasite clearance took as long as five days to accomplish. Parasite clearance was much faster in treatment strategy using the artemether-lumefantrine-primaquine combination than that of chloroquine-primaquine combination. Indeed, artemisinin-based combination therapy (ACT) has been proven to show high efficacy in treating falciparum malaria and non-oroquine-59. TherP. ovale infection with ARDS[P. ovale is capable of causing severe complications and death. For that reason, patients with P. ovale infection should not be regarded as “ultimately benign” and taken lightly, particularly in the non-immune travellers. In view of this, travel history as well as past history of acquiring malaria in patients with fever must be recorded in detail to assist in the diagnosis and management of travellers’ malaria and severe malaria.Severe ovale malaria is not commonly found. There are a few reports of ith ARDS-12, and ith ARDS. Fatal oP. ovale infections with different pathological progress were reported. Plasmodium ovale is potentially capable of causing severe complications, if not death. In view of the differences between these two cases, complete clinical history of malaria patient, especially the travel history and history of malaria exposure are vital for successful treatment. Monitoring of respiratory and renal function of malaria patients, regardless of the species of malaria parasites involved is important during the course of hospital admission. In addition, ACT such as Riamet® should be applied to malaria patients regardless of the species of aetiological agents for prompt and efficient treatment.Two imported cases of The authors declared that they have no competing interests.WCL, YLL, and RM collected, analysed and interpreted the data. LHT, AK and SFSO collected blood samples, conducted clinical diagnoses and treatment intervention. FWC conducted and processed molecular diagnoses. MYF constructed and analysed phylogenetic tree. WCL, YLL, LHT and RM arranged the data, conceptualized and prepared the manuscript. All authors read and approved the final manuscript.


Regular anti-malarial therapy in pregnancy, a pillar of malaria control, may affect malaria immunity, with therapeutic implications in regions of reducing transmission.Plasma antibodies to leading vaccine candidate merozoite antigens and opsonizing antibodies to endothelial-binding and placental-binding infected erythrocytes were quantified in pregnant Melanesian women receiving sulfadoxine-pyrimethamine (SP) with chloroquine taken once, or three courses of SP with azithromycin.p < 0.0001). In contrast, median levels of opsonizing antibodies did not change, although levels for some individuals changed significantly. In multivariate analysis, the malaria prevention regimen did not influence antibody levels.Malaria prevalence was low. Between enrolment and delivery, antibodies to recombinant antigens declined in both groups (Different preventive anti-malarial chemotherapy regimens used during pregnancy had limited impact on malarial-immunity in a low-transmission region of Papua New Guinea.NCT01136850 Plasmodium falciparum infection, and malaria in pregnancy (MiP) increases the risks for mother and baby [Pregnant women are susceptible to and baby . Antibodand baby , 3. Inteand baby , but couand baby . CurrentEthics approval was obtained from the PNG Institute of Medical Research’s Institutional Review Board (08.15), the PNG Medical Research Advisory Council  and the Human Research Ethics Committee of Melbourne Health . Women provided written informed consent.N = 304), control arm, or up to three courses of SP and azithromycin (AZ) (N = 277), intervention arm, and followed to delivery. Women were given an ITN, if available, and usage was recorded. Paired plasma samples collected at enrolment and delivery were assayed for P. falciparum antibodies.Pregnant women were recruited for a malaria prevention trial in Madang, PNG, and plasma samples from a subset of these women were used in this study . ParticiP. falciparum infection  was determined by light microscopy (LMS) and quantitative polymerase chain reaction (qPCR) of peripheral blood films, from placental impression , and by examination of placental histology. Placental malaria was classified as acute, chronic or past infection [Presence of nfection .P. falciparum lines CS2  and E8B-ICAM , and THP-1 monocyte-like cells, were cultured as described [The laboratory-adapted P. falciparum antigens and measles haemagglutinin protein by enzyme-linked immunosorbent assay (ELISA) as described [Samples were assayed for immunoglobulin G (IgG) antibodies to recombinant escribed . In briew/v) paraformaldehyde. The cells were acquired using a HyperCyt® CyAn flow cytometer (Beckman Coulter). Data are represented as percentage of THP-1 cells containing ingested IEs. For all assays, plasma from six adults with no history of malaria exposure was included. Discordant samples were re-run, using published rules [The level of opsonizing IgG antibody was determined as before . In brieed rules .To assess changes during pregnancy, the mean antibody levels from paired samples collected at enrolment and delivery were compared, and the same rules were applied as described . In brieχ2 tests. Multiple linear regression analysis was performed to determine associations between continuous and categorical variables on antibody, and adjusted analyses included treatment arms, maternal characteristics, and interaction variables.The main interest of analysis is whether preventive therapies during pregnancy had an impact on development of pregnancy related anti-malarial antibodies. Data were analysed with Stata version 13.0 (StataCorp) and GraphPad Prism v5 . Differences in population mean ranks of paired continuous non-parametric variables, antibody responses, were evaluated using the Wilcoxon signed-rank test. Categorical variables were compared using the n = 304, SP-AZ n = 277) were similar between groups. Most women receiving SP-AZ (82 %) had three courses. The prevalence of malaria infection was low at enrolment and delivery (Table Participant characteristics (SP-CQ ry Table .Table 1Sp > 0.05). There was a significant decline in median level of antibodies against schizont extract, MSP2, MSP3 and PfRh2 between enrolment and delivery  in both treatment arms. Antibody to measles haemagglutinin (control) showed a similar significant decline in both treatment arms . In contrast to decline in antibodies to recombinant antigens (above), median levels of opsonizing antibodies to both placental-binding and-endothelial-binding IEs did not change significantly between enrolment and delivery in either treatment arm , and in opsonizing antibody to endothelial-binding IEs . Interestingly, significantly more women on SP-AZ than SP-CQ experienced an increase in opsonizing antibody to placental-binding IEs  proteins increased during pregnancy and declined after delivery, suggesting that these antibody responses are transient in the absence of infection [There are many different assays for measuring potentially protective anti-malarial antibody. In Ghana, antibodies to recombinant placental-binding nfection . The lacnfection . In Ghannfection , similarThe relative stability of functional opsonizing antibodies even with low force of infection, compared with IgG directed to recombinant antigens, makes their levels a potential measure of longer lasting immunity to MiP. Whether functional properties of antibodies to IEs, including opsonizing activity and inhibition of placental binding  are the Anti-malarial prevention strategies may affect the development of pregnancy-associated malaria immunity , 13, butIn contrast to antibody to endothelial-binding IEs, levels of opsonizing antibodies to placental-binding IEs were more likely to increase or decrease (rather than remaining constant) in the SP-AZ arm compared with the SP-CQ arm, consistent with previous observations . This reIn the adjusted analyses, factors influencing malarial antibody levels at delivery did not vary between the treatment arms. Increasing gravidity is associated with higher opsonizing antibodies to IEs consistent with gravidity-dependent acquisition of pregnancy-specific immunity . Rural wThe current study may have underestimated potential benefits of prevention. First, the low malaria prevalence may have reduced the power to observe any potential impact of different treatment arms on the acquisition of pregnancy-associated malaria immunity. Second, although high ITN coverage may reduce the need for intensive IPTp and, consequently, reduce selection pressure on the parasites , 16, higIn conclusion, IPTp with multi-doses of SP-AZ or a single-dose SP-CQ had similar impacts on pregnancy-associated malaria immunity, possibly due to the low prevalence of infection and extensive ITN coverage observed. The decline in merozoite and schizont antibodies observed between enrolment and delivery supports this. This study also demonstrated an apparent maintenance of functional opsonizing antibodies, thought to be important for prevention of morbidity, which deserves further evaluation in other regions and in the context of other preventive measures to elucidate the optimum policy in preventing MiP in such regions.


Plasmodium falciparum represents 99 %. Prompt diagnosis by light microscopy (LM) and rapid diagnostic tests (RTDs) is a key element in the national strategy of malaria control. The heterogeneous epidemiology of malaria in the country necessitates the field evaluation of the current diagnostic strategies, especially RDTs. Thus, the present study aimed to evaluate LM and an RDT, combining both P. falciparum histidine-rich protein-2 (PfHRP-2) and Plasmodium lactate dehydrogenase (pLDH), for falciparum malaria diagnosis and survey in a malaria-endemic area during the transmission season against nested polymerase chain reaction (PCR) as the reference method.Malaria is a public health threat in Yemen, with 149,451 cases being reported in 2013. Of these, PfHRP-2/pLDH RDT. Positive samples (160) and randomly selected negative samples (52) by both RDT and LM were further analysed using 18S rRNA-based nested PCR.A household-based, cross-sectional malaria survey was conducted in Mawza District, a malaria-endemic area in Taiz governorate. A total of 488 participants were screened using LM and P. falciparum were found among patients who reported fever in the 48 h prior to the survey by LM and PfHRP-2/pLDH RDT, respectively.The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the RDT were 96.0 % : 90.9–98.3), 56.0 % (95 % CI: 44.7–66.8), 76.3 % (95 % CI: 69.0–82.3), and 90.4 % (95 % CI: 78.8–96.8), respectively. On the other hand, LM showed sensitivity of 37.6 % (95 % CI: 29.6–46.3), specificity of 97.6 % (95 % CI: 91.7–99.7), PPV of 95.9 % (95 % CI: 86.3–98.9), and NPV of 51.3 % (95 % CI: 43.2–59.2). The sensitivity of LM dropped to 8.5 % for detecting asymptomatic malaria. Malaria prevalence was 32.8 %  with the RDT compared with 10.7 %  with LM. Among asymptomatic malaria individuals, LM and RDT-based prevalence rates were 1.6 and 25.6 %, respectively. However, rates of 88.2 and 94.1 % of infection with PfHRP-2/pLDH RDT shows high sensitivity for the survey of falciparum malaria even for asymptomatic malaria cases. Although the RDT had high sensitivity, its high false-positivity rate limits its utility as a single diagnostic tool for clinical diagnosis of malaria. On the other hand, low sensitivity of LM indicates that a high proportion of malaria cases is missed, underestimating the true prevalence of malaria in the community. Higher NPV of PfHRP-2/pLDH RDT than LM can give a straightforward exclusion of malaria among febrile patients, helping to avoid unnecessary presumptive treatments.The  Plasmodium falciparum being the predominant species responsible for almost 99 % of cases. It is estimated that 43 % of the population are at high risk and a total of 63,484 microscopy-confirmed and 39,294 rapid diagnostic test (RDT)-confirmed cases were reported in 2013 [Malaria is a public health threat causing morbidity and mortality in Yemen, with P. falciparum histidine-rich protein-2 (PfHRP-2) have the highest and most consistent detection rate [Plasmodium lactate dehydrogenase (pLDH) detects all Plasmodium species and is usually combined with PfHRP-2 for malaria screening in areas endemic with multiple species [PfHRP-2-based RDTs against LM as the ‘gold standard’ [PfHRP-2/pLDH RDT for malaria diagnosis against PCR as the reference method during the transmission season in a malaria-endemic area in Taiz governorate.Light microscopy (LM) is still the cornerstone of malaria diagnosis in Yemen, especially in hospitals. However, LM has low sensitivity for detection of low parasite densities, is time-consuming and requires skilled technicians and good reagents , 3. Therion rate . In cont species , 7. The  species . Althoug species , which itandard’ , 10. It tandard’ –13. ThisThe present study is a cross-sectional study in Mawza District, which is a malaria-endemic area located in Taiz governorate, south of Yemen Fig. . Its tot® 3MM filter papers, air-dried and kept in separate plastic bags at room temperature until DNA extraction.The protocol of the study was approved by the Ethical Committee of the University of Science and Technology, Yemen. This study recruited 488 participants through a household-based survey in the period from October 2013 to April 2014. All family members, of all age groups and both sexes, were invited to participate on a voluntary basis from randomly selected houses in the study area. Signed or thumb-printed informed consent was taken from the participants or their guardians before sample collection and after a clear explanation of the study objectives. Data on the age of participants, history of anti-malarial drug intake 1 week prior to the study, and history of fever 48 h before the study were collected using a pre-designed questionnaire. Whole blood samples (about 5 ml) were collected in EDTA tubes from all participants that were tested for malaria by using both RDT and LM. Haemoglobin was measured using Sysmex KX-21N Hematology Analyzer . RDT-positive participants were treated according to the national malaria treatment policy of the Ministry of Health and Population, Yemen. For molecular investigation, blood spots were blotted onto WhatmannThick and thin blood films (one slide per sample) were prepared and stained with Giemsa for approximately 20 min according to standard procedures. Thick films were examined using a light microscope by a qualified laboratory technician for a minimum of 100 high-magnification fields before being recorded as negative for malaria parasites. These films were then blindly examined by an independent malaria microscopist for the confirmation of the first examination. A third blinded examination was conducted for those films having discordant results between the first and second examiners. The results for the third examination were regarded as final. All microscopists in this study were trained by the NMCP. Parasite density per μL of blood was estimated for each sample by counting the number of parasites against 200 white blood cells (WBC), assuming a standard mean WBC count of 8000/μL blood . Samples® Malaria Antigen Pf/Pan test, product code: SD05FK63  that combines the detection of PfHRP-2 and pLDH was used based on the WHO recommendations as being one of the ten top-performing RDTs [The SD Biolineing RDTs . Test ki® Blood and Tissue Kit  according to the manufacturer’s instructions and kept at −20 °C until used. Genus- and species-specific nested PCR assays based on the 18S rRNA gene were used to detect and identify P. falciparum [2, 200 nM of each primer and 1 U of Taq polymerase. The cycling conditions for primary PCR were as follows: an initial denaturation step at 94 °C for 4 min, then 35 cycles at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. Secondary PCR used similar cycling conditions except that the annealing temperature was increased to 58 °C. PCR products were separated by electrophoresis on 1.5 % agarose gel and stained with SYBR® Safe DNA Gel Stain .Genomic DNA was extracted from dried blood spots by using DNeasyKc) was used to assess agreement between results obtained by two different tests [Kc = 0.01–0.20), fair (Kc = 0.21–0.40), moderate (Kc = 0.41–0.60), substantial (Kc = 0.61–0.8), or almost perfect (Kc = 0.81–1) [Data obtained were entered and analysed using the Statistical Package for Social Sciences (SPSS) version 20.0 . The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the RDT and LM were calculated against the nested PCR with their 95 % confidence intervals (CIs). Cohen’s kappa coefficient . The prevalence was four times higher among children <10 years old with the RDT than with LM. Asymptomatic falciparum malaria among the study population was found to be 1.6 % with LM compared with 25.6 % with the RDT. However, rates of 88.2 and 94.1 % of infection with P. falciparum were found among patients who reported fever in the 48 h prior to the survey by LM and PfHRP-2/pLDH RDT, respectively . About 10.5 % (51/488) of participants had self-reported fever. The majority of participants  were anaemic, having haemoglobin levels less than 11 g/dL. LM detected falciparum malaria among 10.7 %  of patients. Of them, 9.6 % (5/52) had a low parasitaemia, 32.7 % (17/52) had a moderate parasitaemia, 42.3 % (22/52) had a high parasitaemia and 15.4 % (8/52) showed only gametocytes. On the other hand, the overall prevalence of PfHRP-2/pLDH RDT detected all LM-positive cases of falciparum malaria, irrespective of parasite density. Of the LM-negative samples, 24.6 % (107/435) were RDT-positive. RDT and LM showed a fair agreement  for the detection of P. falciparum among all participants. However, the two tests had a substantial agreement  for detecting the infection among febrile patients (Table Table PfHRP-2/pLDH RDT had sensitivity of 96.0 % (95 % CI: 90.9–98.3) and specificity of 56.0 % (95 % CI: 44.7–66.8), PPV of 76.3 % (95 % CI: 69.0–82.3), and NPV of 90.4 % (95 % CI: 78.8–96.8). The two types of tests showed a moderate degree of agreement . In addition, the RDT maintained its high sensitivity for the detection of P. falciparum among children <10 years old, asymptomatic participants and those with history of anti-malarial drug intake. However, it showed low specificity, which dropped to about 30 % among people with history of anti-malarial drug intake . A reduction in LM sensitivity was observed among children <10 years old, asymptomatic participants and those with history of anti-malarial drug intake. Although LM showed high sensitivity (93.5 %) for detecting symptomatic malaria, such sensitivity dropped to 8.5 % in case of asymptomatic malaria. On the other hand, LM maintained its high specificity for the detection of P. falciparum , specificity of 97.6 % (95 % CI: 91.7–99.7), PPV of 37.6 % (95 % CI: 29.6–46.3), and NPV of 51.3 % (95 % CI: 43.2–59.2). The two types of tests showed a fair degree of agreement  among afebrile participants, indicating its utility in active case detection. This could help in strategies for reducing malaria transmission by identifying asymptomatic carriers and their subsequent treatment. Similar findings of higher RDT sensitivity have been reported previously [PfHRP-2/pLDH RDT showed a higher NPV than LM (90.4 vs 51.3 %) during the peak seasonal transmission of malaria. This is advantageous for the definite exclusion of malaria among patients, and the avoiding of unnecessary presumptive treatments. Given that the NPV is 100.0 % for the RDT and 40.0 % for LM among febrile patients, RDT-negative results for patients experiencing fever will be straightforward and will rationalize the prescription of anti-malarial drugs. Similarly, a very recent study [The good performance of the eviously . The WHOnt study  recommenP. falciparum missed by LM can be detected by RDTs because of the release of PfHRP-2 by parasites and its circulation in the blood [PfHRP-2 have been suggested as a better alternative to LM in areas of low-density parasitaemia and their false positives compared with LM have been confirmed by PCR to be cases below the threshold detection of LM [The superiority of RDTs compared with LM could be explained by the fact that sequestered he blood , 24. Meaon of LM . In addion of LM .PfHRP-2/pLDH RDT in the present study is in contrast to the high specificity (96.1 %) recorded for the CareStart™ HRP-2 RDT tested in an earlier study [PfHRP2-based RDTs for falciparum malaria diagnosis among febrile patients in Malawi, where specificity of 39–68 % was reported [PfHRP-2/pLDH RDT but negative by the PCR reference method. Although these were considered as false-positive results compared to nested PCR as the reference method, unperceived factors contributing to the inhibition of PCR could not be ruled out. Moreover, PCR false-negativity has been documented in the literature compared to LM [P. falciparum using PfHRP-2-based RDTs reported from Congo [PfHRP-2-based RDT false positivity and its relatively low PPV could be attributed to the persistence of PfHRP-2 antigenaemia in the blood circulation for 4–5 weeks after parasite clearance with successful treatment [PfHRP-2/pLDH RDT in the present study. Previous studies showed a negative correlation between the specificity of RDTs and malaria prevalence [PfHRP-2/pLDH RDTs can lead to overdiagnosis and subsequent overtreatment, which may contribute to the emergence and spread of drug resistance [P. falciparum-specific LDH-based RDTs should be evaluated for screening of falciparum malaria in Yemen. This may help avoid the drawback of PfHRP-2 RDTs resulting from persistent antigenaemia in blood after treatment and cure, minimizing the false positivity rate to reasonable and acceptable levels. However, PfHRP2 positivity in the absence of P. falciparum-specific LDH or pan-specific LDH does not necessarily mean a false-positive result due to persistent antigenaemia [In contrast, LM had higher overall specificity than RDT (97.6 vs 56 %) compared with the reference method. LM is still the gold standard for species identification and detecting the severity of malaria by quantifying parasitaemia and for differentiation of transmissible stages from those responsible for clinical disease . Low speer study , which creported . It is ned to LM , 28–30. om Congo , 32 and om Congo . The PfHreatment , 33, 34.evalence , 35–37. sistance . Therefogenaemia , it is nP. falciparum was three times higher when using PfHRP-2/pLDH RDT compared with LM and 16 times higher among asymptomatic patients. In this context, Mappin et al. [Differences in sensitivity and specificity reflect on the estimation of falciparum malaria prevalence in the country, particularly among asymptomatic patients. In the present study, the overall prevalence of n et al.  reportedn et al. , 43. Altn et al. . Howevern et al.  and Angon et al. . OverallPfHRP-2/pLDH RDT tested in the present study showed a better performance than LM in field survey for malaria, even in asymptomatic cases. It showed a moderate degree of agreement with nested PCR, with a high sensitivity. A major drawback of the RDT is that the high false-positivity rate limits its use as an independent diagnostic tool for malaria to avoid unnecessary overtreatment. However, its negative results totally exclude falciparum malaria among febrile patients as indicated by its 100 % NPV. This can have public health implications in educating healthcare providers and patients in endemic areas to perform RDTs in all cases of fever to exclude falciparum malaria before prescribing or taking anti-malarial drugs. The low sensitivity of LM indicates that a high proportion of malaria cases is missed, leading to an underestimation of the true malaria prevalence in the community. However, LM remains indispensable to species identification, differentiation of gametocytes from asexual stages and the assessment of the severity of the disease. Furthermore, it is still the gold standard for the diagnosis of symptomatic malaria. RDTs should be further investigated as rapid malaria-excluding diagnostics among febrile patients in endemic areas.The


Plasmodium falciparum. In this study, we aimed to evaluate the immunogenicity of HRP 2 exon II and the efficacy of novel monoclonal antibodies (mAbs) against HRP 2 for Point-of-Care Test (POCT).Malaria presents a considerable threat to public health. Histidine-rich protein 2 (HRP 2) is the major protein released into human blood upon infection by E. coli and used to immunize mice for mAb production. Two IgG1 mAbs (1A5 and 1C10) with high affinity, specificity and sensitivity for both native and recombinant HRP 2 were selected after fusion of mouse spleen with myeloma cells. The affinity constant of 1A5 and 1C10 were 7.15 and 4.91 × 10-7 L/mol, respectively. Subsequently, an immunochromatograhic assay was used for screening of clinical samples in endemic regions of China and Myanmar.The recombinant protein was expressed in soluble form in The immunochromatographic test retrospectively showed an overall sensitivity of 99.07%, and specificity of 100%. Sensitivity at parasite densities < 200, 200–2000, and > 2000 parasites/μL was 87.5, 98.7, and 100%, respectively.These results suggest that HRP 2 exon II contains immunogenic sites similar to those of the native antigen and can be used for the development of mAbs suitable for malaria diagnosis in endemic communities. Plasmodium species, is one of the oldest and largest health challenges affecting 40% of the world population [P. falciparum infection, the most lethal malarial plasmodia responsible for the cerebral form of the disease. Approximately 86% malaria deaths are children under 5 years of age [Plasmodium falciparum prevalence rates in most parts of China and Myanmar have been brought under control, very high transmission rates still remain in certain regional communities [Plasmodium related cases with increased antimalarial drug resistance in Southeast Asia, including China [Malaria, a disease caused by pulation . There ws of age -4. TheseP. falciparum in these regions has resulted in drug resistant species, pointing to the need for improved diagnosis and monitoring of the disease. The total eradication of malaria is one of the urgent aims of the United Nations Millennium Development Goals. The methods recommended by WHO for P. falciparum diagnosis include microscopic examination, immunological tests, and PCR methods [Inaccurate and ineffective diagnosis of  methods -9.T3 initiative in 2012, all malaria-endemic and donor countries should ensure that every suspected case of malaria is tested and treated, [Plasmodium antigens have been described, of which Plasmodium falciparum specific histidine-rich protein 2 (HRP 2), and Plasmodium lactate dehydrogenase (pLDH) and aldolase are common to all four Plasmodium species [Since the launch of WHO’s treated,  requirin species . The PfH species .The most convenient technique for clinical diagnosis of malaria is the use of rapid diagnostic test kits, which depends on the use of mAbs against the HRP 2 antigen. These tests are particularly important since they can be used in field diagnostics  to screen large populations without the requirement of trained laboratory personnel or equipment. Several tests targeting HRP 2 are available, with various specificities, sensitivities, and temperature tolerances, illustrating the difficulties and challenges facing current RDTs . The difIn this study, we aimed to express the HRP 2 exon II antigen retaining the essential epitopes that can serve as immunogens for use in developing mAbs against HRP 2 for POCT immunochromatographic assay, according to guiding principles formulated by the WHO . The resPlasmodium falciparum as shown in Figure Clinical samples n=516) were collected between 2008 and 2012 from patients (6–50 years old) with fever and suspectable for malaria from Henan (n=90) and Yunnan (n=202) provinces of China  and from Myanmar (Burma) region (n=224), where malaria is endemic and is caused mainly by 6 were coDiagnostic Criteria for Malaria issued by the Ministry of Health of the People's Republic of China. Patients were included if they ①came from relatively serious malaria epidemic area and ②suffered from fever of unknown origin. Patients were excluded if they ①suffered from vivax malaria or ②had mixed infections. The samples were confirmed as single malignant malaria infection by microscopic examination of thick blood smear.The diagnosis of malaria follows An expert medical technologist collected approximately 5 mL of blood from adult subjects and 3 mL from minor subjects by venipuncture. Thick and thin blood films were prepared in duplicate using two drops of blood for each sample. The remaining blood was preserved at −20°C in EDTA tube. Thick blood films were stained with 10% Giemsa and microscopic examined by experienced technicians.Positive references provided by WHO were divided into 5 geographical areas, comprising Africa (Benin I and Nigeria XII), the Americas (Santa Lucia), Australia (Papua New Guinea-FC27/A3) and Southeast Asia (Philippines- PH1).Plasmodium falciparum genomic DNA was extracted from clinical blood samples provided by Prof. Hui Liu , using QIAamp DNA Mini Kit . Pair of oligonucleotide primers was designed, complementary to forward and reverse strands of the HRP 2 exon II based on the sequence obtained from GenBank (U69551.1). The forward primer was HRP 2-F: 5׳-TATACATATGAATAATTCCGCATTTAATAATAACT-3׳, and the reverse primer HRP 2-R: 5׳-TACAGTCGACATAGACGTACTTCTTTTCGTAAG-3׳. The forward and reverse primers were designed to contain NdeI and XhoI restriction sites, respectively. Restriction endonucleases (NdeI and XhoI), DNA markers and PCR mix were purchased from Dongsheng Biotech Company , while the T4 DNA Ligation mix was obtained from TaKaRa Biotech Co. Ltd . The HRP 2 gene was cloned into a pET30a vector and transformed into CaCl2 competent E. coli BL21 (DE3) cells [2+ column  using AKTA Purifier and the purified recombinant HRP 2 protein (rHRP 2) fractions visualized on 12% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [3) cells . pET-30aDS-PAGE) . The proSix-to-eight-weeks-old Balb/c mice were purchased from the Animal Laboratory Center of Sun Yat-sen University . All experimental procedures were in compliance with the Guide for Care and Use of Laboratory Animals . All animals were allowed free access to food and water.P. falciparum infected blood samples. SDS-PAGE was carried out on 12% separating and 5% stacking gels, followed by protein transfer to polyvinylidene fluoride (PVDF) membranes. SuperSignal West Pico Chemiluminescent Substrate  was applied for visualization. Determination of antibody affinity was carried out by a noncompetitive ELISA as described by David Beatty [MAbs were produced as described by Kohler and Milstein  and scred Beatty  with litMicrotiter plates were coated with 1 μg/ml of purified rHRP 2 in coating buffer . After overnight incubation at 4°C, plates were rinsed with washing solution  and blocked with 3% bovine serum albumen (BSA) for 2 h. 100 μL hybridoma cell supernatants were added to wells and incubated for 1 h at 37°C. After washing, 100 μL goat anti-mouse IgG-HRP  antibodies were dispensed into each well followed by 1 h incubation at 37°C. The enzymatic reaction was visualized using TMB substrate with hydrogen peroxide and stopped with 2 M Sulphuric acid. Optical density (OD) was measured spectrophotometrically at 450 nm. Samples were considered positive with OD of cell supernatant (P) to negative control (N) ratios greater than 2.1.Purified mAbs were paired, labeled with periodate oxidation and sandwich ELISA was performed as described before . The low2 to form test and control lines, respectively. The prepared nitrocellulose membranes were kept in a dry pack at 30°C and 30% humidity. 1C10 antibody were conjugated to colloidal gold as described by Oliver [2. The strip was assembled by placing 0.7 cm of antibody-gold conjugate glass fiber a few centimeters from the test line and 4 mm wide test strips were cut. Test strip preparation was done in a good manufacturing practice (GMP) workshop.HRP 2 specific mAb 1A5 (1.5 mg/ml) and goat-anti-mouse IgG (2.0 mg/ml) were diluted in 0.05 M carbonate buffer and sprayed on a nitrocellulose membrane at 1.5 and 1 μl/cmy Oliver  and spraPositive control samples provided by WHO were evaluated alongside 516 clinical samples and test results were compared with microscopic examinations. About 5 μL of each sample was applied to the sample site and three drops of dilution buffer added. Test strips were observed for 15 min and results were interpreted as positive when both the control and test lines turned red and negative if only the control line turned red. In cases where the control line did not appear, the results were considered invalid and the assay repeated.Sensitivity of the test equals the ratio of total positives to total number of samples, specificity equals the ratio of total negatives to total number of samples, positive predictive value equals the proportion of the number of positive cases detected among actual positives cases and negative predictive value equals the proportion of the number of negative cases detected in relation to actual negatives.Statistical analysis was performed with SPSS 18.0 software . Data obtained from both microscopic examination and immunochromatographic test were analyzed. Sensitivity and specificity of the assay and how it compares with the standard microscopic examination was analyzed. Agreements between both data were computed by Kappa statistics. Differences were considered statistically significant at p < 0.05.Plasmodium falciparum infections, with no mixed infections detected (n=322) including 60/90 (66.7%) samples from Henan, China, 105/202 (52.0%) samples from Yunnan, China and 157/224 (70.1%) from Myanmar. Negative Plasmodium falciparum blood samplesn=194) were also assessed by thick and thin smear microscopic examinations and confirmed to be Plasmodium falciparum negative, including 30/90 (33.3%) samples from Henan, China, 97/202 (48.0%) samples from Yunnan, China and 67/224 (29.9%) from Myanmar.Microscopic inspection showed that most samples were positive for only E. coli BL21 to express soluble recombinant HRP 2 exon II after IPTG induction. Hybridomas were developed using mouse spleen cells screened by indirect ELISA. After three rounds of limiting dilution, 5 stable cell lines producing mAb against PfHRP 2 were selected. mAbs from the 5 cell lines were paired in a sandwich ELISA  and 1A5/1C10 pair was chosen for further analysis and detection of clinical samples. The titer values for mAb 1A5 and 1C10 were greater than 106 and their affinity constants were 7.15 and 4.91 × 10−7 L/mol, respectively. Both mAbs were of the IgG1 isotype. Figure Plasmodium falciparum cell culture supernatants and pLDH antigen. The result showed that mAbs 1A5 and 1C10 recognize recombinant and nature HRP 2 sensitively and spectively.The 831 bp DNA fragment was transformed into All positive references were examined retrospectively with both microscopy and the immunochromatographic assay. Differences in detection levels were noted Figure . The difA total of 516 blood samples collected from 6–50 years old patients were examined. The newly developed test kit detected 164 of the 165 positive samples originating from China (sensitivity of 99.39%), with no false positives in the 127 negative samples examined. In samples collected in Myanmar area, a total of 157 positive and 67 negative cases were found, with a sensitivity rate of 98.72%. Three false negatives were recorded. These results are summarized in Table P. falciparum infection [Malaria is a major public health problem worldwide and its total eradication is one of the urgent aims of the United Nations Millennium Development Goals. Hence the use of fast, accurate, and easy on-site detection methods and reagents as monitoring medical tools for early diagnosis and treatment of malaria in regions of high transmission and prevalence is particularly important. However, the performance of existing HRP 2 tests in areas of intense malaria transmission varies by age and prevalence of nfection . The parnfection .E. coli and purified its full amino acid sequence. After administration of the purified rHRP 2 antigen in mice followed by fusion of mouse splenocytes with myeloma cells, two different hybridomas (1A5 and 1C10) producing anti-HRP 2 mAbs were selected and used for the preparation of ascitic fluids in mice. After purification of ascitic fluids by ammonium sulfate precipitation and Protein A column, specificity of 1A5 and 1C10 was assessed by immunoblot: both antibodies exhibited remarkable specificity for detection of both recombinant and native P. falciparum HRP 2 antigen, binding to no other protein in human blood serum.In the current study, we aimed to evaluate the immunogenicity of HRP 2 exon II for use in developing mAbs against HRP 2 for POCT. We have successfully expressed recombinant HRP 2 exon II in −7 and 4.91 × 10−7 (unit) for 1A5 and 1C10, respectively, an indication of the high binding affinity of these two mAbs. The high affinity observed for the expressed exon II region of PfHRP 2 suggests that epitopes are conserved in this region. Therefore, the recombinant PfHRP 2 protein developed in this study can serve as immunogen to facilitate the production of high affinity and high specificity mAbs for use in clinical diagnosis of malaria caused by P. falciparum. The genetic diversity of HRP 2 affects the performance of the HRP 2-based RDT [A noncompetitive ELISA method was used in the determination of antibody affinity. We found association constant (Ka) values of 7.15 x 10ased RDT .Microscopic examination of both thick and thin blood smears is a traditional detection method and hence considered the gold standard for malaria diagnosis. However, due to its complicated operation and the requirement of experienced laboratory technicians, field application is limited. POCT, a new concept for rapid diagnosis of clinical samples developed in recent years is very crucial. POCT is based on small, portable, easy to operate, fast, accurate, and effective devices for on-site monitoring of infections. Sensitivity of HRP detection kits is affected by several factors including the quality of mAbs used, immunogenic response of B cells to the different epitopes of HRP 2 protein , and theThe lateral immunochromatographic assay was developed with the two HRP 2 specific mAbs and used for testing of blood samples collected from high endemic regions in China and Myanmar. There was a strong agreement between the immunochromatographic test results and microscopic examination among the 322 positive and 194 negative samples tested (K=0.9955 at p < 0.001). The observed sensitivity and specificity were 99.07 and 100%, respectively (Table P. falciparum infections. Because of the limited supply of mAbs, it is very likely that most HRP 2-based RDTs use similar or identical antibodies. Existing HRP 2-based RDTs are known to yield false-positive results [P. falciparum, resulting in saved lives and reduced occurrence of parasite resistance.mAbs with acceptable sensitivity, specificity and high affinity for the PfHRP 2 antigen are prerequisite for accurate diagnosis of  results . TherefoThe Plasmodium falciparum HRP 2 exon II contains immunogenic sites similar to those on the native antigen. These immunogenic sites can effectively be utilised for the development of highly sensitive and specific mAbs to facilitate malaria diagnosis in endemic communities.