Plasmodium falciparum and Plasmodium vivax in Queensland, Australia, from 2006 to 2016. All LFT results from malaria diagnosis until 28 days after diagnosis were collected with demographic and clinical information to describe longitudinal changes. The timing of peak LFT elevations was classified as early (0–3 days), delayed (4–11 days), or late (12–28 days) with respect to the day of diagnosis. Among 861 cases with LFT evaluated, an elevated bilirubin level was identified in 12.4% (N = 107/861), whereas elevated alanine transaminase (ALT) and aspartate transaminase levels were observed in 15.1% (N = 130/861) and 14.8% (N = 127/861) of cases, respectively. All peak bilirubin results occurred in the early period, whereas ALT elevations were biphasic, with elevations in the early and delayed periods, with 35.4% (N = 46/130) of cases delayed. Univariate and paired stepwise logistic regression analyses were performed to investigate factors associated with the incidence and timing of transaminase elevation. A raised ALT level at diagnosis was strongly associated with the timing of transaminase elevation. No other demographic, parasitic, or treatment factors were associated. Liver function test abnormalities are likely an inherent although variable aspect of human malaria, and individual-specific factors may confer susceptibility to hepatocyte injury.Liver dysfunction has long been recognized as a clinical feature of malaria. We have observed delayed elevation in the transaminase portion of liver function tests (LFTs) after treatment in some participants undergoing induced blood stage malaria infection. We sought to determine whether similar LFT elevations occur after naturally acquired infection. We performed a retrospective audit of confirmed cases of  Abnormalities in liver function have been described but relatively little investigated compared with other aspects of clinical malaria. Reporting of malaria-associated liver injury in the literature is heterogeneous and primarily focused on biochemical measurement of liver function tests (LFTs), such as bilirubin and the transaminases alanine transaminase (ALT) and aspartate transaminase (AST). There are few thorough longitudinal descriptions of malaria-associated liver injury.7 Other Plasmodium species have been associated with a relatively lower rate of jaundice compared with falciparum.9 Mild elevations of transaminases are common, although more significant elevations associated with multiorgan dysfunction have also been reported.14 The syndrome of “malarial hepatopathy” has been recently proposed, being defined as a bilirubin level > 2.5 times upper limit of normal (ULN) with associated transaminase elevation > 3 × ULN (where ALT is considered the more liver-specific enzyme).19 Although the clinical significance of malarial hepatopathy has not been fully elucidated, it represents an attempt to further describe malaria-associated liver injury and has been associated with more severe disease and other organ dysfunction.1 The relationship of malarial hepatopathy to antimalarial treatment needs to be evaluated to differentiate it from clinically significant drug-induced liver injury meeting Hy’s Law, which has a similar definition.20 Malarial hepatopathy has been reported in 2.6–45% of all malaria cases and up to 87.5% of cases presenting with clinical jaundice.22 Kupffer cell hyperplasia, hemozoin loading, and monocyte infiltration are the most frequently reported histological findings in malaria-associated liver injury; all seem to resolve after antimalarial treatment.27Surveys of falciparum malaria have reported lower rates of jaundice at presentation in endemic populations (2.6–5.3% of cases) compared with epidemic malaria (11.5–62% of cases).16Although the incidence of abnormal LFTs has been described in multiple studies, the temporal pattern of LFT abnormalities has been less explored. Available data indicate that LFT abnormalities due to malaria typically resolve after 1–2 weeks compared with 6–8 weeks after acute hepatitis A, B, or C.31 In a small retrospective comparative European study, asymptomatic transaminase elevation was observed in returned travelers hospitalized for malaria after treatment with the artemisinin-based combination therapy artemether–lumefantrine. The authors postulated that the rapid parasiticidal effect of the antimalarial treatment may promote accelerated hepatic heme loading and oxidative stress.28 Liver injury through this proposed mechanism is not exactly an adverse drug reaction but rather may be influenced by factors, such as parasite burden, rate of clearance, and host heme metabolism. In prospective trials using the induced blood stage malaria (IBSM) model in healthy malaria-naive participants, asymptomatic moderate (2.5–5.0 × ULN) to more rarely severe (> 5.1 × ULN) elevations of transaminase enzymes have been reported in a subset of participants. These isolated transaminase elevations are associated with an ALT/AST ratio > 1 and a normal bilirubin level, peaking 4–12 days after treatment, and have been reported following multiple antimalarial compounds in both falciparum and vivax malaria models.30 Similar elevations have been observed after sporozoite malaria challenge.32 Delayed transaminase elevation may be attributed to the antimalarial compound, the use of acetaminophen for symptom relief,33 the malaria parasite, or host factors associated with parasite clearance. Although the relative contribution of each of these factors is uncertain, it is important to understand their relative contribution, given the implications that unexplained transaminase elevation may have on antimalarial drug development.Peaks in transaminase levels after treatment have also been recently reported; these could represent a distinct type of malaria-associated liver injury.Plasmodium falciparum and Plasmodium vivax infection in Queensland, Australia. In this report, we describe the incidence, degree, and timing of abnormal LFTs in a population of patients with naturally acquired malaria infection. Cases with LFT outcomes of interest were specifically investigated to describe the temporal relationship between antimalarial treatment and transaminase elevation. We also analyzed factors that may contribute to transaminase elevation, specifically the timing of peak elevation after treatment.To further characterize the spectrum of malaria-associated liver stress/injury, we performed a retrospective audit of cases of P. falciparum and P. vivax malaria on the Pathology Queensland electronic database  between January 1, 2006 and June 1, 2016, an interval that covers the period of electronic pathology record keeping in the State. Inpatient and outpatient data were included. Duplicate results, defined as any malaria positive test in the same individual for the same Plasmodium species collected within 7 days, were excluded. The reference case for inclusion was considered the earliest test collected at a Queensland Health facility. Plasmodium species coinfections were included. Plasmodium falciparum was considered the reference infection unless P. vivax monoinfection was diagnosed within 7 days earlier.We included patients of any age with microscopy-confirmed cases of Supplemental Table 1).Data on demographics , parasitemia (by microscopy at the time of diagnosis), and LFTs  from the day of malaria diagnosis (Day 0) to 28 days after diagnosis were collected from the Pathology Queensland database for all cases. Where multiple results were found for a single day, the earliest result was recorded. All LFT results were converted to multiples of the ULN to allow for comparison between age and gender groups (34 Mild (Grade 1) elevations of LFTs were not considered clinically significant in the context of documented malaria . Malarial hepatopathy was defined as bilirubin > 2.5 × ULN with a concurrent ALT > 3 × ULN.1 The peak LFT measurement and day after malaria diagnosis in which it was measured were used to determine the severity and temporal pattern, respectively. We grouped LFT results into three time periods: early (Day 0 to Day 3), delayed (Day 4 to Day 11), and late (Day 12 to Day 28). Time periods were based on the timing of delayed transaminase elevation observed in healthy nonimmune participants in IBSM trials.30Liver function tests were classified using a modified World Health Organization (WHO) toxicity grade system (Grades 0 to 4), which is used to assess adverse events in clinical trials .34 Mild Early, delayed, and late transaminase elevations were defined as having a moderate, severe, or very severe grade peak ALT result within the appropriate time period. The reference normal group was defined as cases with serial ALT measurements < 2 × ULN across contiguous time periods. This cutoff was chosen to increase the size of this group for comparison. An electronic chart audit and a review of Public Health records were conducted to identify the geographical region where malaria was acquired and the type and timing of treatment regimen, where this information was documented.Plasmodium species, presence of other infections, and treatment regimen were compared between groups to identify possible contributing factors to transaminase elevation. Data on the use of potentially hepatotoxic medications were not available.Treatment regimen was classified as artemisinin-containing or nonartemisinin-containing. The timing of treatment initiation with respect to the day of diagnosis was recorded to determine the treatment delay. Other infections potentially contributing to abnormal LFTs were identified in Pathology Queensland records. This was defined as any positive acute phase testing for dengue, other arboviruses, herpes viruses, viral hepatitis, and other infections  occurring within 28 days after malaria diagnosis. Log-transformed ULN data for total bilirubin, ALT, AST and the ratio of ALT/AST at diagnosis, demographics, region of acquisition, log-transformed parasitemia at diagnosis, P < 0.05 was considered statistically significant. A paired stepwise logistic regression model was used in an exploratory multivariate analysis of all clinical/demographic factors available to determine, which were independently predictive of transaminase outcome. Likelihood ratios (LRs) were calculated for variables considered predictive of outcome by the model (P < 0.05). No adjustments were made. GraphPad Prism 7.0  was used for descriptive statistics and univariate analysis. JMP Pro 13.2  was used for the paired stepwise logistic regression analysis.Descriptive statistics are presented as mean with standard deviation (SD), median with interquartile range (IQR), or percentage. Cases were grouped by outcomes: early, delayed, and late transaminase elevations or serial normal transaminase results. Parasitemia and LFT × ULN results were log transformed to normalize data distribution. Assessment of normality of distribution was undertaken using the D’Agostino test. All groups were compared using the chi-squared test, Kruskal–Wallis test, or one-way analysis of variance to compare available clinical/demographic data between groups. A It is not possible to identify the true frequency of malaria-associated LFT elevations from this retrospective cohort, as many patients with malaria infection did not undergo LFT measurement. The exact timing of peak LFT elevation is not known for many cases because daily testing was not performed. Cases with early LFT abnormalities or other complications may have undergone more frequent monitoring and be overrepresented in the study population.This study was approved by the Royal Brisbane and Women’s Hospital Human Research Ethics Committee .A total of 1,477 individual cases were assessed . Of thesP. falciparum  and an older age at diagnosis , but no difference in gender distribution or parasitemia at presentation.Compared with patients who did not have LFT measurement, those who had LFTs collected had a significantly higher rate of infection with N = 107/861) of all cases with LFT measurements (N = 21/861) of all cases.The level of bilirubin was elevated > 2.5 × ULN in 12.4%  of all cases with LFT measurements. New cases were identified up to Day 9 after diagnosis. Moderate, severe, and very severe peak elevations were observed in 76/130, 41/130, and 13/130 cases, respectively (N = 84/130) and 35.4% (N = 46/130) in the delayed period. No cases had peak LFT elevations in the late period. Aspartate transaminase elevation was identified at rates similar to ALT elevation, although the temporal distribution of peak AST elevation appeared to favor a slightly earlier onset .An elevated ALT level > 3 × ULN was identified in 15.1% (ectively . The peaer onset . TransamThe median (IQR) ALT/AST ratio of all LFT measurements on Day 0 was 1.0 (0.7 to 1.2). The ALT/AST ratio progressively increased to consistently > 1 after Day 4. There was no difference in the ALT/AST ratio based on infective species.P. falciparum than in P. vivax cases  or mildly elevated (N = 43/861) LFTs on the day of diagnosis that subsequently went on to develop moderate or greater abnormalities. Liver function test results by day of collection for these cases are presented in Supplemental Figure 1.Of all cases with LFTs undertaken, there were 54 cases with normal . Furthermore, the ALT/AST ratio at diagnosis was also higher in the early transaminase elevation group (P = 0.003). Region of malaria acquisition (P < 0.001) and positive testing for another infection potentially contributing to LFT abnormalities (P = 0.005) were the only nontransaminase indices that differed between groups. Data on region of acquisition was more readily available in the normal transaminase group, which had fewer “unknown” category results than the other groups. Positive testing for other infections was also most common in the normal transaminase group. All groups demonstrated a similar age and gender distribution. Furthermore, P. falciparum was the most common infective species across all groups, and parasitemia at presentation was similar. The median delay to treatment was 1 day after diagnosis, and the majority of cases received an artemisinin-containing regimen . Region of acquisition , lower log bilirubin level on the day of diagnosis , higher rates of P. vivax infection , and female gender  were also associated with early transaminase elevation compared with the normal transaminases group. Delayed transaminase elevation was associated with a higher log ALT level on the day of diagnosis compared with normal transaminases . Region of acquisition was also different between these groups . Comparison of the delayed and early transaminase groups found that higher log ALT on the day of diagnosis  and positive testing for other infections  were associated with early transaminase elevation.Multivariate analysis by paired comparison of normal transaminases and early transaminase elevation unsurprisingly found log ALT on the day of diagnosis had an extremely strong association with early transaminase elevation . We have also identified a relatively large proportion of cases with elevated transaminases after diagnosis and treatment that has not been well described previously in the naturally acquired malaria literature. Although elevations in bilirubin levels appear to peak and resolve soon after treatment, peak transaminase elevations may not occur simultaneously. This may represent malaria-associated liver effects after hemolysis, which could explain the progressive increase in the ALT/AST ratio observed with time for all cases, as the hemolysis-related AST level decreases while the hepatocyte-specific ALT level increases.In this audit, we have identified rates of elevated bilirubin and malarial hepatopathy consistent with other reports.35 In this study, delayed ALT elevation peaking 4–11 days after diagnosis was common. This could represent an underdescribed burden of malaria-associated liver injury, especially given that the frequency of LFT measurements is reduced with time since diagnosis, and LFTs are frequently not performed serially if normal or only mildly elevated at diagnosis.Analysis of factors potentially contributing to the presence and timing of transaminase elevation predictably demonstrated that the ALT level on the day of diagnosis had the greatest association with the timing of transaminase elevation. This is unsurprising for cases with LFTs peaking in the first 3 days and is an inherent limitation of using this variable. Interestingly, those who experienced delayed elevation had a higher ALT level on the day of diagnosis compared with the normal transaminases group, suggesting that hepatic injury was already underway. Given that treatment was instituted soon after diagnosis, this delayed transaminase elevation may suggest continued hepatocyte injury in the postparasite clearance period. Serum ALT typically responds to hepatocyte injury within hours after insult rather than days.28Plasmodium falciparum infection was associated with a higher frequency of abnormal LFTs overall. However, when the three time period groups were compared, the proportion of P. falciparum cases compared with P. vivax cases was only different between the normal and early transaminase elevation groups. Interestingly, infection with another organism that may cause liver injury such as dengue or viral hepatitis was more common in the early transaminase elevation group compared with the delayed transaminase group, which may have contributed to earlier peak hepatocyte injury. Based on the available data, the normal transaminases group had the highest absolute rate of coinfection, which is not biologically plausible. Differences between groups in region of malaria acquisition are similarly difficult to interpret, given the low number of cases, high number of categories, and discrepancies in data availability between comparison groups but could be related to either population or parasite strain-related differences.Treatment regimen and the timing of institution of treatment after diagnosis were not associated with the timing of peak transaminase elevation. This is in contrast to other studies associating artemisinin-containing treatment regimens, and presumably more rapid parasite clearance, with LFT elevations.40Although this analysis was limited, there do not seem to be clear demographic or clinical differences between groups that are predictive of liver injury. We hypothesize that other individual-specific factors may confer susceptibility to hepatocyte injury. This may include host ability to manage reactive oxygen species generated by heme breakdown after erythrocyte recycling in the liver and the degree of proinflammatory immune response to infection. In this population, the bilirubin level at the time of diagnosis was only different between normal and early transaminase elevation groups, which may support the hypothesis that the burden of hemolysis alone does not modulate the timing of transaminase elevation. This is supported by mechanistic studies of malaria-associated liver injury in mouse models, which demonstrate that heme-mediated oxidative damage is promoted by tumor necrosis factor (TNF) in a synergistic fashion.41 These elevations occurred in the same time period as the delayed transaminase elevations described in this study, which followed a range of antimalarial treatments, and are of a similar severity. If delayed transaminase elevation is truly part of the spectrum of malaria-associated liver injury as we propose, it is clear that assigning causality in the trial setting will continue to be challenging. This problem will exist at least until either individual predisposing factors for malaria-associated liver injury are better understood or biomarkers to distinguish between drug-induced liver injury and malarial hepatopathy are identified.It is clear that the pattern of abnormal LFTs is variable in malaria infection, ranging from acute hepatopathy associated with jaundice and poor prognosis, to asymptomatic transaminase elevation. Differentiating malaria-associated liver injury from drug-induced liver injury is important both at the bedside and in the context of antimalarial drug development. This study has found that elevations in bilirubin levels and malarial hepatopathy occur exclusively in the few days after diagnosis and treatment, which may be a useful feature to help differentiate malaria associated changes from potentially more delayed significant drug-induced liver injury meeting Hy’s Law. The identification of delayed transaminase elevation in this study provides more of a challenge, given that this likely forms part of the normal pathophysiology of disease but varies between individuals. This adds an extra layer of complexity to interpreting safety data from clinical trials undertaken for antimalarial drug development. For example, in field studies of pyronaridine–artesunate in uncomplicated malaria, mild transient transaminase elevations were reported on Days 3 and 7 after treatment, which were attributed to direct low-level drug toxicity.By including all cases of falciparum and vivax malaria irrespective of initial LFT results and hospital or community setting, this study population is broader than that typically reported. Nonetheless, the retrospective nature of this audit means that the datasets are incomplete, which is a major limitation of this report. Furthermore, although it is assumed that the study population is largely malaria naive, given that Australia is malaria-free, this could not be confirmed from the available data. The use of potentially hepatotoxic medications such as acetaminophen often used in malaria patients, and which may affect LFT results, was not recorded in this study. Many confirmed cases of malaria did not have LFTs for review. In those who had LFTs collected, there is likely a selection bias toward sampling in more severe syndromes and serial testing in those with already abnormal results. This may explain why those with any LFTs collected were older and had a higher proportion of falciparum malaria compared with those with no LFTs. Although this may result in overestimation of the rate of abnormal LFTs in early malarial illness, it may also result in underestimation of delayed transaminase elevations. The absolute rate of delayed transaminase elevation in malaria cannot be determined from this analysis, as the delayed time period was used to find cases. A prospective study with LFT sampling after completion of treatment is required to measure the incidence of this phenomenon. Genotyping heme oxygenase 1—the enzyme that catalyzes heme degradation—in patients, and measuring circulating TNF levels at diagnosis and after treatment, could be used to test the hypothesis that individual heme metabolism and TNF-associated inflammatory response may confer susceptibility to malaria-associated liver injury.Liver function test abnormalities occur commonly in persons infected with various plasmodia and are likely an inherent although variable aspect of human malaria. Elevated transaminase levels due to malaria are transient, may be delayed beyond antimalarial treatment, and may not occur simultaneously with bilirubin changes. No clinical or demographic factors are clearly related to transaminase elevation; however, extrapolation of mouse-model studies may suggest that individual variation in inflammatory response and the efficiency of heme breakdown could play a role in transaminase elevation.Supplemental Figures and Tables


Malaria is one of the killer diseases in Ethiopia and it is still the first leading cause of death in health facilities. However, there is no information yet regarding the trends of malaria prevalence at health institution and particularly at Felegehiwot referral Hospital. Hence, knowing the trends of malaria prevalence at each health facilities is essential to design appropriate interventions. Therefore, the present study addressed the above gaps.Plasmodium falciparum and Plasmodium vivax accounted for 397 (53%) and 331 (45%), respectively. Age groups >20 year (p < 0.02) and males (p < 0.025) were significantly affected.Overall, 14,750 blood films were diagnosed for malaria. Of these, 740 (5%) were confirmed with microscope. P. falciparum was predominant as compared to P. vivax. Hence, it needs close monitoring and intervention measures for control activities.In conclusion,  Plasmodium falciparum  accounts for 55% and Plasmodium vivax (P. vivax) for 45% of the cases. Reports also depicted that clinical malaria accounts for 10–40% of all out patient consultations and morbidity among children under 5 years in age being 10–20% [In Ethiopia, according to the ministry of health report, P. falciparum resulted in a change for treatment. As a result, artemether/lumefantrine (Coartem ®) (AL) was adopted [P. vivax even if, its resistance has not been yet known. Above all, self-treatment is common practice and this will result in development of drug resistance for the commonly prescribed drugs.In Ethiopia, four major intervention strategies are still applied to combat malaria . Moreove adopted , 4. On tOn top of these, different reports depicted confused results on decrement of malaria. Moreover, knowing the trends of malaria prevalence at each health institution is essential to design appropriate interventions. Furthermore, there is no information yet regarding the trends of malaria prevalence at health institution and particularly at Felegehiwot referral Hospital. Therefore, the present study addressed the above gaps.A retrospective study was conducted to determine a 5 year trend analysis of malaria prevalence with in the catchment areas of Felegehiwot referral Hospital by reviewing malaria blood film malaria reports from 2010 to 2014. Bahir Dar city is situated on the Southern shore of Lake Tana, the source of the Blue Nile in what was previously the Gojjam province and now the Amhara National Regional State. The city is located approximately 578 km Northwest of Addis Ababa, having an elevation of 1840 meters above sea level. Based on the 2007 Census conducted by the Central Statistical Agency of Ethiopia, it has a total population of 221,991, of whom 108,456 are males and 113,535 females [Plasmodium species and season were collected on suspected malaria cases requested from 2010 to 2014 at Felegehiwot referral Hospital.5 year data on malaria cases were obtained from Felegehiwot referral Hospital. Laboratory technologists who examined blood films have 7–10 years of experience. To maintain the validity of this examination, a well-prepared and well-stained thin and thick blood films are used as the gold standard in confirming the presence of the malaria parasite as WHO protocol. The staining techniques and blood film examination for malaria parasite detection were conducted according to a standard operating procedure (SOP) stated on manual for the laboratory diagnosis of malaria  in each Data was analysed using Statistical Package for Social Sciences (PSS) version 20. Descriptive statistics such as line and bar graphs were used to show the trends of malaria in terms of season and year, respectively. Chi-square test was used to compare the trends of malaria, male and female and also between different age groups. Finally, p < 0.05 was considered as statistical significant.P. falciparum accounted for 397 (2.7%) and P. vivax for 331 (2.2%). Plasmodium species infection had been observed more in males 406 (2.8%) than females 334 (2.3%). The difference was statistical significant (p < 0.025) (Table For the last 5 years 2010–2014), a total of 14,750 blood films were diagnosed for malaria at Felegehiwot referral Hospital. Of these, 7, 329 (49.7%) were males and 7, 421 (50.3%) were females with the median age of 23. The overall prevalence of microscopy—confirmed malaria parasitaemia was 740 (5%). Of these, 0–2014, aP. falciparum was highest 58 (14.6%) as compared to P. vivax 39 (12%) and mixed infections 3 (2.5%). In age groups from 5 to 9 and 10 to 15, infection with P. vivax was dominant. In age groups from 16 to 20 and >20, there was a sharp increment in infection with both P. falciparum and P. vivax. Increments in mixed infections were observed from age groups 10–15 through >20 year. Statistical significant association was observed between age groups and infection with P. falciparum and P. vivax (p < 0.02) . This finding was smaller than a retrospective study done in Northwest Ethiopia  and SoutAnopheles mosquito bites.In this study, the overall prevalence of malaria in males 406 2.8%) was higher than females 334 (2.3%). This result was in line with the study done in Southwest Ethiopia [% was higP. falciparum 397 (2.7%) and P. vivax 331 (2.2%) in the present study was lower than studies reported from other parts of the country [The prevalence of  country , 10. ThiIn our study, the age groups, >20 years 411 (55.5%) were highly affected followed by 16–20 years old 135 (18.2%) but from 10 to 15 years old 39 (5.2%) were the least affected. This study coincides with a study done in Northwest , but difP. falciparum throughout the year revealed that it seems stable transmission. The reason might be since Felegehiwot Hospital is a referral Hospital; new cases could be referred from malaria stable transmission areas. The highest prevalence was observed from September to December followed by May to June. This result was in agreement with the monthly trends of malaria transmission stated in ministry of health [Months and seasonality have a direct role in the transmission of malaria. In our study, the prevalence of f health . This moP. vivax infection was observed compared to P. falciparum with the peak at 2012 which seems epidemic. This was in line with a report in Southwest Ethiopia [P. vivax [Plasmodium species in the study area.According to this study, there were irregular occurrences of cases each year for the last 5 years. From 2011 to 2012, more Ethiopia . This miThe 5 year prevalence of malaria in our study area was lower than the National Ministry of Health. Therefore, more work will be done using PCR and RDT tests having more sensitivity.


Plasmodium falciparum was 0.3%, 4.3%, 4.0%, and 7.8% by light microscopy, rapid diagnostic test (RDT), conventional polymerase chain reaction (cPCR), and multiplexed real-time PCR (RT-PCR), respectively, and Plasmodium vivax prevalence was 0% by all detection methods. In Laiza, of 385 asymptomatic participants, 2.3%, 4.4%, and 12.2% were positive for P. vivax by microscopy, cPCR, and RT-PCR, respectively, and 2.3% were P. falciparum-positive only by RT-PCR. Of 34 symptomatic participants in Laiza, 32.4% were P. vivax-positive by all detection methods. Factors associated with infection included gender , and young age group . Although the sensitivity of microscopy was adequate to detect symptomatic infections, it missed the vast majority (86.5%) of asymptomatic infections. Although molecular detection methods had no advantage over standard microscopy or RDT diagnosis for clinically apparent infections, malaria elimination along the Myanmar–China border will likely require highly sensitive surveillance tools to identify asymptomatic infections and guide targeted screen-and-treat interventions.Malaria infections may be symptomatic, leading to treatment, or “asymptomatic,” typically detected through active surveillance, and not leading to treatment. Malaria elimination may require purging both types of infection. Using detection methods with different sensitivities, we conducted a cross-sectional study in two rural communities located along the border between China’s Yunnan Province and Myanmar’s Shan and Kachin States, to estimate the prevalence of asymptomatic and symptomatic malaria. In Mong Pawk, all infections detected were asymptomatic, and the prevalence of  Plasmodium falciparum malaria, in 2015 the World Health Organization (WHO) recommended launching a regional campaign to eliminate P. falciparum malaria from the Greater Mekong Subregion, which includes Myanmar, Thailand, Cambodia, Vietnam, Laos, and China’s Yunnan Province.1 China developed malaria elimination strategies to interrupt local malaria transmission in the entire nation except for the Yunnan–Myanmar border by 2015, and to be completely malaria-free by 2020.2 The malaria burden in Yunnan decreased dramatically from 2006 to 2013, but local transmission has persisted, particularly in Menglian and Yingjiang counties that are located adjacent to China’s border with Myanmar’s Shan and Kachin States, respectively. Both P. falciparum and Plasmodium vivax are present in this region, with P. vivax accounting for more than 60% of reported cases in 2014 .3,4 Transmission is unstable with frequent outbreaks, and up to 50% of malaria cases in Yunnan Province are suspected to be imported from across the long borders with Laos and Myanmar, which have the highest malaria burdens in the region.5In hopes of preventing the spread of artemisinin-resistant 6 which may serve as a reservoir and contribute to ongoing malaria transmission in the face of interventions targeting clinically apparent infections.7,8 Although standard diagnostic tests such as light microscopy or rapid diagnostic tests (RDTs) can adequately identify symptomatic infections with high-enough parasite densities, the sensitivity of these tests may be insufficient to detect asymptomatic infections, which can have much lower densities, particularly in low transmission settings such as those found in southeast Asia.9 Most studies of asymptomatic malaria infection have used microscopy or RDTs, which have lower limits of detection (LoD) of about 100 parasites/µL (or 105/mL), or more recently, conventional nested polymerase chain reaction (cPCR), with the LoD ranging between 1,000 and 5,000 parasites/mL.10 In contrast, a high-volume ultrasensitive PCR assay, using 2 mL of venous blood, has an LoD of 22 parasites/mL11 and another finger-prick ultrasensitive PCR assay’s LoD is < 16 parasites/mL,12 several 1,000-fold lower than the LoD of standard diagnostic methods. The epidemiology of subclinical malaria has recently been described for sites in Thailand, Cambodia, and Vietnam, where these infections are being targeted by mass drug administration.9,13 To expand geographic breadth of our understanding of the epidemiology of subclinical malaria, we conducted a cross-sectional field study to estimate the prevalence of malaria infection in the populations residing at two study sites located on the border between the Yunnan Province of China and northeastern Myanmar’s Shan and Kachin States. We identified malaria infection from a very small volume of blood (40 µL) collected on filter papers using multiplexed real-time PCR (RT-PCR), which has an LoD between those of conventional and ultrasensitive PCR, as well as cPCR, and standard light microscopy and/or RDT.Malaria infection can be asymptomatic, or, more accurately, “subclinical,” because subtle symptoms and chronic health effects may occur but not lead to clinical diagnosis and treatment. Malaria elimination may require eradicating both clinically symptomatic as well as these “silent” infections,Cross-sectional community surveys were conducted in June–August 2014 in Mong Pawk and Laiza, two small communities on the Myanmar–China border . Mong PaAll willing individuals who provided written informed consent were included in the study. Exclusion criteria included age less than 6 months, and history of taking any antimalarial or antimicrobial drugs in the 14 days before enrollment.Plasmodium vivax malaria was treated with a total dose of 1,200 mg chloroquine over 3 days, followed by eight daily doses of primaquine 180 mg. Plasmodium falciparum malaria was treated with eight tablets of fixed-dose combination of dihydroartemisinin and piperaquine (40 mg/320 mg) for 3 days. Blood collected onto Whatman3MMfilter paper  were labeled with a unique identification number, air-dried, and individually placed in plastic bags with desiccant, and stored at a room temperature until laboratory analysis.Participants were recruited by word of mouth . During a specified study period of 3 months, participants were invited and recruited at a small study unit set up at a community center, on a first-come-first-serve basis. Special effort such as recruitment in the evening and on holidays was made to maximize the chance of including participants of all ages, genders, and occupations. Written informed consent was obtained from all adult participants, along with assent and adult guardian consent for children aged < 18 years. Demographic data, including age, gender, and occupation, and medication and travel history were collected. The presence of any symptoms of illness was recorded and axillary temperature taken. Blood was collected from a fingertip or earlobe, for thick and thin smears and RDT (which was available only at the Mong Pawk site) for immediate diagnosis of malaria, and onto filter paper for later PCR analysis. Participants who tested positive for malaria by RDT or microscopy were referred to a malaria clinic affiliated with the study team to receive standard treatment, following the Chinese national malaria treatment guidelines. P.f/P.v; SD Standard Diagnostics, Inc., Suwon, Korea; lot number 145113, expiration date September 9, 2015) were used at the Mong Pawk site, following the manufacturer’s instructions. Targeted antigens in the RDT were P. falciparum-specific HRP-2 and P. vivax-specific pLDH. The previously reported sensitivities of this RDT in patients with clinical malaria were 99.7% and 95.5% for P. falciparum and for P. vivax, respectively,14 but are unknown in detecting malaria in asymptomatic individuals.Thick and thin blood smears were prepared using Giemsa stain and independently examined under a microscope by two experienced microscopists. One hundred fields of a thick smear were screened before reporting. If the thick smear was positive, 200 fields of a thin smear were screened for species identification. Ten percent of randomly selected negative slides and all positive slides were cross-checked by an independent microscopist as external quality control. All microscopists were certificated as level 1 by WHO monitors. RDTs  The second amplification was accomplished by using 2 µL of the first PCR product as a template under the same 20-µL reaction mixture conditions. The reaction conditions were as follows: 95°C for 5 minutes, 30 cycles of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 2 minutes, followed by a single 60°C elongation step for 10 minutes. Amplified products were visualized in 2% agarose gels stained with ethidium bromide. The expected sizes of the P. falciparum and P. vivax PCR products were 206 and 121 base pair, respectively.DNA was extracted from dried blood spots using the QIAamp 96 DNA Blood Kit  following the manufacturer’s instructions and the total elution DNA volume for each sample was 100 μL. The cPCR was based on primers targeted to the 18S ribosomal RNA gene described previously.16 with fluorophores and concentration modified for this study. The primer and probe with the respective concentrations for each reaction are shown in Plasmodium species of each sample was determined with species-specific forward primers, Plasmo2, and species-specific probes. The reaction was performed in a single tube with distinct fluorophores for each probe. The threshold for P. falciparum was 0.1 relative fluorescence units (RFU) at 40 cycles, whereas for P. vivax it was set to be 0.04 RFU at 40 cycles. A negative control consisting of the DNA from a negative sample was included in the test panel along with 3D7 P. falciparum and P. vivax genomic DNA, both from the Malaria Research and Reference Reagent Resource, as positive controls.RT-PCR was performed under universal conditions  with the ABI 7500 Real Time system. The reaction was performed with a final volume of 25 µL containing 5 µL of DNA, 12.5 µL of TaqMan universal master mix , primers, and probes. The primers and probes sequences were published previously,P. falciparum-positive by RT-PCR were genotyped for the Kelch 13 artemisinin resistance marker. A nested PCR amplification method performed following previously reported protocols17 with minor modifications. PCR products were purified using filter plates . The product was labeled with Big Dye Terminator v3.1 Reaction Kit (Applied Biosystems) before ethanol precipitation and capillary electrophoresis on an ABI 3730XL automatic sequencer as recommended by the manufacturer’s instructions.Samples testing 2 test. The independent effect of the risk factors on malaria infection was determined using multivariate logistic regression analysis.The data were processed, described, and analyzed using Microsoft Excel 2010  and SAS 9.3 . The prevalence of malaria infection was calculated as the proportion of infection-positive participants divided by the number of successfully tested participants using each study method . Potential risk factors for malaria infection evaluated included study site, age , gender, and travel to areas known or suspected to be malaria endemic. The prevalence of asymptomatic malaria infection within each of these risk categories was calculated and assessed using a χThe study protocol was independently reviewed and approved by the Institutional Review Boards of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, and the University of Maryland, Baltimore, MD.A total of 765 participants (346 from Mong Pawk and 419 from Laiza) were enrolled. Of those, 191 (55.2%) and 224 (53.6%) were female in Mong Pawk and Laiza, respectively. The median (range) age of participants was 23.8 years (0.5–80 years) and 23.5 years (0.8–81 years) in the Mong Pawk and Laiza sites, respectively. The age distribution was similar between the two sites . All parP. falciparum and P. vivax infection, detected by different study methods, is shown in P. falciparum—no symptomatic infections and no P. vivax infections were detected at this site. Among 346 participants, 1, 15, 14, and 27 infections were identified by microscopy, RDT, cPCR, and RT-PCR, respectively. The prevalence of asymptomatic malaria infection was 7.8% by RT-PCR. Compared with RT-PCR, microscopy missed 96%, RDT missed 44%, and cPCR missed 48% of infections. In Laiza, of 385 asymptomatic participants, 9, 17, and 47 were positive for P. vivax by microscopy, cPCR, and RT-PCR, respectively, and 9 were positive for P. falciparum infections only by RT-PCR. Among 34 participants who had fever or other symptoms suggestive of acute clinical malaria, none tested positive for P. falciparum by any of the study methods, and 11 were positive for P. vivax consistently by microscopy, cPCR and real-time PCR. Of the 11 patients (72.7%), eight were of age between 5 and 40 years. RDT testing was not done at this study site. A map of the study site and prevalence of malaria is shown in The prevalence of P. vivax infections, the concordance between microscopy, cPCR, and RT-PCR was 100%, with 11 samples being positive for P. vivax by all three tests. For asymptomatic P. vivax, only nine of 47 RT-PCR-positive infections were microscopy-positive, whereas 8 of these nine (89%) microscopy-positive infections were positive by both cPCR and RT-PCR. All 17 cPCR-positive asymptomatic P. vivax infections were also positive by RT-PCR, whereas cPCR missed 30 of 47 (64%) RT-PCR-positive infections.For symptomatic P. falciparum infections, all of which were asymptomatic, microscopy missed all but one of the 14 cPCR-positive and 27 RT-PCR-positive infections. RDTs missed five of 14 cPCR-positive and 18 of 27 RT-PCR-positive infections, but only nine of the 15 RDT-positive infections were positive by cPCR and RT-PCR. All cPCR-positive P. falciparum infections were also positive by RT-PCR, whereas cPCR missed 22 of 36 (61%) RT-PCR-positive P. falciparum infections.For falciparum-positive samples detected by RT-PCR were genotyped by capillary sequencing, and 12 of these were successfully sequenced. All of them were found to contain wild-type K13, with no detectable polymorphisms known or suspected to be associated with artemisinin resistance.A total of 36 P = 0.0012), 1.76 times higher in males compared with females , and 2.71 times higher in the 5–17 age group compared with individuals < 5 years of age . The log odds of malaria infection in the other age groups did not differ significantly from that in individuals aged < 5 years. Travel history was not significantly associated with any malaria infection after adjusting for study site. This is likely because of the fact that all but one of the individuals with a travel history had a symptomatic infection, and all of the symptomatic infections were from the Laiza study site.Study site, gender, age, and travel history were assessed as risk factors for malaria infection determined by RT-PCR . In an uThis cross-sectional survey revealed the presence of reservoirs of asymptomatic malaria in otherwise generally healthy plantation workers and refugees living in remote rural areas along the border between China’s Yunnan Province and Myanmar’s Shan and Kachin States. The size of this “silent” reservoir was substantially underestimated by standard RDT or microscopy in this study, which detected malaria infection using cPCR and RT-PCR, in order of increasing sensitivity.P. falciparum infections were negative by both cPCR and RT-PCR. Possible explanations for this discordance include false-positive RDTs or unreported recently treated infections with residual antigenemia after parasite nucleic acids had disappeared.In participants with acute clinical malaria at the Laiza site, malaria was detected uniformly by microscopy, cPCR, and RT-PCR (RDT was not available in this study site), suggesting that the sensitivity of standard diagnostic tests is adequate when parasite density is high enough to cause clinically recognizable malaria illness in this setting. The higher sensitivity and lower LoDs of molecular testing thus offered no advantage for diagnosing clinical malaria at this site. However, in participants with subclinical malaria, the majority of the infections detected by RT-PCR were missed by light microscopy, RDT, or even cPCR, likely because of low parasite densities that fell below the LoDs of the less sensitive methods. Unexpectedly, in Mong Pawk, more than one-third of RDT-positive asymptomatic 9,18,19 Our findings suggested that even conventional or standard PCR, which is commonly used for both research and surveillance, may underestimate the true prevalence of subclinical malaria. We used a low volume of dried blood spot (∼14 μL), which may have further limited the sensitivity of our molecular detection methods. The examination of a high volume (∼2 mL) of blood has been used to measure the prevalence of subclinical malaria along the Thailand–Myanmar border.11 We have recently developed an ultrasensitive multiplexed reverse transcriptase PCR method that achieves similarly ultralow LoDs using finger-stick samples and requires no processing or cold chain.12 We are adapting these methods for use on dried blood spots, aiming to achieve LoDs lower than those of the RT-PCR we used here, ideally as low as obtained with venous and capillary samples.Others have reported that the prevalence of malaria infections was significantly higher when a highly sensitive real-time PCR method was used.11,13,19,20 The contribution of low-density infections that can be detected only by sensitive molecular methods to the risk of transmission is unknown, but presumably the parasites have evolved to persist in this form for their survival benefit. Even if the risk of transmission from low-density infections is low at one time point, it is plausible that these infections fluctuate in their density and ability to generate infectious gametocytes. Limited data from Cambodia suggest that persistent asymptomatic infection is more frequent in vivax (35%) than falciparum (13%) malaria.21 Further research is needed to assess the extent to which these very low density P. falciparum and P. vivax infections contribute to the risk of clinical malaria and/or of transmission to inform elimination strategies in the Greater Mekong Subregion. Understanding these risks is essential for weighing the risks and benefits of interventions needed for malaria elimination in populations with infections that are undetectable by RDT and seen only with ultrasensitive tests.Although the point-of-care testing ability offered by microscopy or RDTs is essential for malaria case management, the inability of these tests detect the majority of subclinical infections poses a serious challenge to the success of malaria elimination planned in Greater Mekong Subregion, where malaria transmission is low and where the majority of infections are likely to be present in this silent form.P. falciparum infections—all asymptomatic—were found. Standard RDT surveillance would have completely failed to detect a significant malaria burden at this site. In contrast, refugees in Laiza, in northeastern Kachin State, had predominantly P. vivax infections, and a significant minority were experiencing clinical symptoms of malaria. The border of Yunnan Province with Shan and Kachin States is one of the last remaining frontiers of malaria endemicity in China, which is striving for malaria elimination by 2020. The presence of a border-spanning asymptomatic reservoir, acting as the primary driving force for continued transmission, complicates the task of elimination, and the predominance of P. vivax in some areas poses the additional challenge or treating relapsing malaria.Malaria epidemiology was different at the two sites. Among rubber plantation workers in Mong Pawk, located in Myanmar’s eastern Shan State, only 9,22 This unexpected finding suggests that either children are going to where the vectors are , or vectors are present where children spend their time (in or near the home or school). At the Mong Pawk plantation site, there was no difference in malaria prevalence between young children and adolescents (data not shown), who spend most of their time at home or school. But the risk was higher in young children compared with adolescents in the Laiza refugee camp. We speculate that daytime malaria transmission may be occurring near the primary school attended by the younger children. Entomological investigation is needed to confirm this speculation.Interestingly, our study found a significantly higher malaria prevalence among children. This was surprising in light of the fact that clinical malaria in this region is generally recognized to be more common in young adults with occupational exposure to forest-biting mosquitoes.23 where the risk of resurgence is elevated by the presence of multidrug resistance. Myanmar accounts for approximately one-fifth of the region’s population, more than half of the malaria cases and approximately three-quarters of malaria deaths in this region.24 Cross-border migration between the Yunnan, China, and Myanmar is thought to be the major source of introduction of P. falciparum malaria into southern China.4 Malaria outbreaks and re-introduction of malaria in areas previously known to be malaria-free have been frequent in these border areas.25 The majority of malaria infections in the Myanmar–China border areas have been determined to be imported from Myanmar, based on travel history. Malaria importation occurs as a result of human activities involving border trade, businesses, border development, and mass population movement due to political instability.26,27Malaria remains an important infectious disease in the Greater Mekong Subregion,As we approach the goal of elimination, malaria parasites can be expected to go to ground, residing at low densities in people and at low prevalence in remote rural locations, especially along border areas that are often beyond the reach of routine health care and surveillance. Molecular epidemiological or seroepidemiological tools that go beyond simply measuring prevalence to estimate parasite migration patterns and identify transmission sources and sinks could further improve our understanding of asymptomatic reservoirs and transmission dynamics in this region, and enhance prospects for elimination.


Anopheles vector bites and parasite prevalence.Seroepidemiology can provide evidence for temporal changes in malaria transmission and is an important tool to evaluate the effectiveness of control interventions. During the early 2000s, Vanuatu experienced an acute increase in malaria incidence due to a lapse in funding for vector control. After the distribution of subsidised insecticide-treated nets (ITNs) resumed in 2003, malaria incidence decreased in the subsequent years. This study was conducted to find the serological evidence supporting the impact of ITN on exposure to Plasmodium falciparum  and three Plasmodium vivax  antigens, as well as the Anopheles-specific salivary antigen gSG6, were detected by ELISA. Age-specific seroprevalence was analysed using a reverse catalytic modelling approach to estimate seroconversion rates (SCRs).On Ambae Island, blood samples were collected from 231 and 282 individuals in 2003 and 2007, respectively. Parasite prevalence was determined by microscopy. Antibodies to three P < 0.001) from 19.0% in 2003 to 3.2% in 2007, with a shift from P. falciparum predominance to P. falciparum-P. vivax co-dominance. Significant (P < 0.001) decreases were observed in seroprevalence to all three P. falciparum antigens but only two of three P. vivax antigens , consistent with the more pronounced decrease in P. falciparum prevalence. Seroprevalence to gSG6 also decreased significantly (P < 0.001), suggesting that reduced exposure to vector bites was important to the decrease in parasite prevalence between 2003 and 2007. Analyses of age-specific seroprevalence showed a three-fold decrease in P. falciparum transmission, but the evidence for the decrease in P. vivax transmission was less clear.Parasite rate decreased significantly .Serological markers pointed to the effectiveness of ITNs in reducing malaria prevalence on Ambae Island between 2003 and 2007. The recombinant gSG6 antigen originally developed to indicate exposure to the Afrotropical vector  In recent years, analyses of antibody responses to one or more malaria-specific antigens have been widely used to assess transmission intensity –3, tempoPlasmodium falciparum and Plasmodium vivax, with some rare cases of Plasmodium malariae [P. falciparum than P. vivax [Anopheles farauti (s.s.), a member of the Anopheles punctulatus complex [Located at the margin of malaria transmission, Vanuatu is an archipelago of more than 80 islands in the South Pacific. Malaria is endemic on most of the nation’s 68 inhabited islands . The majmalariae , 11. TheP. vivax , 12. The complex .Several large-scale control programmes have been implemented in Vanuatu over the past decades. In 1988, insecticide-treated nets (ITNs) were introduced; by 1992 ITN coverage had reached 27% of Vanuatu’s population . The shaP. falciparum and P. vivax, exposure to vector mosquito bites was also evaluated, with the aim to determine the effect of vector control on parasite prevalence. The three parasite antigens used in this study vary in immunogenicity. The Plasmodium crude schizont extract (SE) is multi-antigenic and has greater sensitivity to detect low residual transmission and changes in transmission intensity [19 (MSP-119) exhibits moderate immunogenicity and has been useful for estimating malaria transmission in populations across a gradient of transmission intensity [19 [An. gambiae salivary gland antigen (gSG6) showed strong immunogenicity among African populations in previous studies [Anopheles abundance [This study reports on the parasitological and sero-epidemiological results from samples collected on Ambae Island in 2003 and 2007. In addition to responses to antigens from ntensity . The blo studies , 19. Thebundance .2) is in Penama Province  and June 2007 (n = 282). Futuna Island (11 km2) is in Tafea Province  and were included in the analyses of parasite prevalence and vector exposure only.Ambae Island , who also recorded the name of each participant as he/she enrolled in the survey. Gender and age were recorded for each participant. Axillary body temperature was determined using a digital thermometer . Fever was defined as a temperature exceeding 37.5 °C. Spleen size was assessed in children aged 12 years and younger according to Hackett’s method by one investigator only (AK). Blood samples were obtained for microscopic examination of malaria infections. Two spots of blood (70 μl each) were collected on Whatman 31ET Chr filter paper . The blood spots were air-dried and stored in plastic bags at ambient temperature in the field and later at -20 °C in our laboratories until processing. This study was approved by the Ministry of Health in Vanuatu and by the Committee on the Ethics of Human Research of Karolinska Institutet in Sweden.P. falciparum gametocytes was recorded.Thin and thick blood smears were stained with 3% Giemsa solution for 30 min and examined under oil immersion  by experienced microscopists. Blood smears were defined as negative if no parasites were found after examining 100 high power microscopy fields. For all positive samples, malaria species were identified, and the presence of A blood spot (3 mm in diameter) was punched from each sample and antibodies were eluted in reconstitution buffer in 0.5 ml deep well plates  as described previously . The rec19), and recombinant apical membrane antigen-1 (AMA-1) for P. falciparum  and P. vivax  as described previously [P. falciparum and P. vivax antigens were prepared as previously described [19 at 0.5 μg/ml. The plates were washed in PBS with 0.05% Tween 20 (PBS/T) and blocked using 1% (w/v) skimmed milk (Sigma-Aldrich) in PBS/T for three hours. After washing, 50 μl of reconstituted serum were added in duplicate. The final antigen dilutions were 1:1000 for SE and MSP-119, and 1:2000 for AMA-1. In addition, four blank wells and a five-fold dilution series of an African hyper-immune serum pool (n = 12) were added per plate. The plates were washed and 50 μl of horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG antibody  was added at a dilution of 1:15,000 in PBS/T and incubated for 3 h. After further series of washes, antibody responses were detected after development with 100 μl of the substrate solution 3, 3', 5, 5'-Tetramethylbenzidine (TMB)  for 15 min. The reaction was stopped with 50 μl of 2 M H2SO4. The optical density was read using Multiskan Go ELISA reader  at 450 nm.All sera from Ambae were tested for IgG antibodies by indirect quantitative enzyme-linked immunosorbent assay (ELISA) to crude schizont extract (SE), the 19 kDa fragment of recombinant merozoite surface antigen-1  using the recombinant An. gambiae salivary gland gSG6 antigen [All samples from Ambae and Futuna were tested for exposure to bites of  antigen . ELISA wpost-hoc tests. Pairwise correlations between ODs of different antibody responses were determined using the Spearman’s rank correlation. Seroprevalence was stratified into yearly age groups and then analysed using a reverse catalytic modelling approach under a binomial sampling assumption, as described elsewhere [Plasmodium was defined as being positive for either or both species-specific antigens (MSP-119 and/or AMA-1). Survey year, gender, age group, fever, and infection status were considered as explanatory variables in the univariate analyses. All variables with a P-value of ≤ 0.05 from the likelihood ratio test in the univariate analyses were included in the multivariate logistic regression model. The variables included in the final (adjusted) model was survey year and age group.Data were double-entered and imported in STATA/SE version 13.1 . Optical density (OD) values were averaged and normalised against values from blank wells to adjust for background reactivity as previously described . Seroposlsewhere , 28. ThiPlasmodium infections were significantly lower in 2007 than in 2003 (Table P < 0.01). In both sampled years, most Plasmodium infections were found in children 10 years and younger  than P. vivax (-5.1%), resulting in a shift in species composition from the predominance of P. falciparum in 2003 to the co-dominance of P. falciparum and P. vivax in 2007. The prevalence of P. falciparum gametocytes also decreased significantly (Fisher’s exact test: P < 0.001) between the sampled years  than that from Ambae in both 2003 and 2007. The prevalence of fever on Futuna was similar to that on Ambae in 2003  decrease on Ambae between 2003 and 2007. No significant difference in gSG6 antibody levels was observed between Ambae in 2007 and Futuna, where Anopheles vectors are absent. Similar to the decrease in anti-gSG6 antibody levels, median antibody levels to all P. falciparum and P. vivax antigens also decreased significantly  on Ambae between the survey years. Significant  differences in median antibody levels were observed among parasite-specific antigens on Ambae in 2007. For P. falciparum, the anti-SE antibody level was significantly (Mann-Whitney U-test: both P < 0.01) higher than the anti-MSP-119 and anti-AMA-1 levels. For P. vivax, the anti-AMA-1 antibody level was significantly (Mann-Whitney U-test: both P < 0.05) higher than the anti-SE and anti-MSP119 levels.Antibody levels as measured in optical densities are shown in Fig. vs PfMSP-119) than between those of orthologous antigens (e.g. PfMSP-119vs PvMSP-119). Weaker correlations were observed between antibody responses to gSG6 and the parasite antigens .Antibody responses were significantly correlated among all antigens, although the strength of correlation varied Table . In geneP. falciparum and P. vivax. Similar to parasite rate and other malaria indices, seroprevalence was significantly higher  in 2003 than in 2007 for all parasite antigens except PvAMA-1. No significant difference was observed in seroprevalence between males and females for any of the antigens tested . For all parasite antigens, the proportion of seropositive individuals significantly increased with age . Seroprevalence to gSG6 is shown in Fig. P < 0.001) decrease in seroprevalence to gSG6 was observed, from 39% in 2003 to 0.7% in 2007. Nevertheless, gSG6 seroprevalence in 2003 was similar among age groups.Figure P. falciparum antigens significantly decreased between 2003 and 2007 on Ambae, as evidenced by the non-overlapping confidence intervals. The resulting SCRs suggest at least a three-fold decrease in P. falciparum transmission on Ambae. For P. vivax, significant decreases in SCRs were observed for SE and MSP-119, but not for AMA-1 between 2003 and 2007. The decreases in SCRs were smaller in magnitude for P. vivax antigens than their counterparts for P. falciparum.The relationship between seroprevalence and age on Ambae was further examined using reversible catalytic conversion models. Seroconversion curves for parasite antigens are shown in Fig. P. falciparum and P. vivax specific antigens, as well as Anopheles gSG6 on Ambae Island (Table ). In the adjusted model, the survey year 2007 was significantly associated with lower seropositivity to all antigens tested. Significant associations were found between age and seropositivity to both P. falciparum and P. vivax antigens. For P. falciparum antigens, the likelihood of seropositivity increased significantly with age, whereas for P. vivax, the increase in seropositivity was significant among adults aged > 20 only. No association was found between age and seropositivity to the vector salivary gSG6 antigen.We performed univariate and multivariate logistic regression analyses to identify factors associated with seropositivity to any nd Table . In theP. falciparum at the national level were observed after 2006 (16). In this study, we examined the parasite prevalence and IgG antibody responses to antigens from the two major Plasmodium species as well as Anopheles vector on Ambae Island in 2003 and 2007, marking the peak of the resurgence and its subsidence, respectively. A significant decrease in parasite prevalence was accompanied by significant decreases in seropositivity to most parasite antigens and the vector salivary antigen, suggesting that reinforced vector control played an important role in the reduction of malaria transmission on Ambae.Following a gap in funding from external donors that negatively impacted ITN coverage, Vanuatu experienced a resurgence of malaria incidence in the first half of the 2000s. In 2003, Vanuatu secured financial support from the Global Fund to resume distribution of subsidised ITNs. Subsequent decreases in slide positivity rate and proportion of cases caused by P. falciparum cases. As ITN was the main intervention tool used during the study period, the shift to P. falciparum-P. vivax co-dominance in 2007 suggests that ITNs were more protective against P. falciparum-infected mosquitoes. Previously in Papua New Guinea, differential ITN protection was partially explained by differences in biting behaviour of An. punctulatus mosquitoes infected with different parasite species, with a higher proportion of P. vivax-infected mosquitoes biting humans earlier in the evening (25). Furthermore, ITNs provide no protection against P. vivax relapse from hypnozoites, which might explain the increase in the proportion of P. vivax cases in our study site as well as others, where overall malaria incidences have declined as a result of effective interventions [The shift in species predominance on Ambae between 2003 and 2007 was consistent with the nation-wide decrease in the proportion of ventions , 29, 30.P. falciparum prevalence between 2003 and 2007 was reflected in the more pronounced reduction in seroprevalence to P. falciparum antigens, especially AMA-1  and P. vivax (-0.239). It should be noted that crude SE contains multiple proteins, of which some are similar between parasite species [P. falciparum and P. vivax.The greater decrease in P. falciparum antigens were higher than the ones for P. vivax antigens, reflecting the more intense transmission and the predominance of the former species on Ambae, especially in 2003 , 70.2% for PfMSP-119 (0.114 to 0.034), and 66.9% for PfAMA-1 (0.127 to 0.042). For P. vivax, the decreases in transmission were more variable: 69.8% for PvSE (0.043 to 0.013), 60.0% for PvMSP-119 (0.020 to 0.008), and 13.3% for PvAMA-1 (0.015 to 0.013). It is unclear why the estimates from PvMSP-119 and PvAMA-1 were drastically different. The difference in SRR, subclass dependent half-life, inherent immunogenicity, and polymorphism or diversity between the two antigens may explain some of the variation in estimates [Seroprevalence reflects cumulative malaria exposure and can be used to estimate transmission intensity in a population. Modelling changes between seroprevalence and age (i.e. SCR) can help evaluate specific interventions in an area. In practice, SCR is calculated by fitting a reversible catalytic model to the age-specific malaria seroprevalence data, taking into account malaria exposure over time . SCR desstimates –33.On Aneityum Island, SCRs decreased by 85.0% (0.04 to 0.006) for PfSE and 93.3% (0.03 to 0.002) for PvSE seven years after the implementation of an integrated malaria elimination programme in 1991 . The obsAn. gambiae [Anopheles species [An. gambiae population from Burkina Faso [Anopheles species [An. gambiae gSG6 shares high degrees of identity with orthologues from the main African , Asian  and European  malaria vectors, whereas a more limited identity (52%) was found with An. farauti . Notably, SG6 is absent in An. albimanus and An. darlingi, members of the subgenus Nyssorhynchus and important malaria vectors in Central and South America. The current study is the first to describe the use of recombinant gSG6 antigen to evaluate exposure to Anopheles bites in the Pacific, where all major malaria vectors belong to the An. punctulatus group [Anopheles farauti (s.s.), the only malaria vector in Vanuatu, is absent on Futuna Island . Accordingly, all participants from Futuna in this study were seronegative to gSG6 . Nevertheless, a recent survey showed lower ITN use on Ambae (68%) than on Aneityum (73%), where elimination-specific effort has successfully halted malaria transmission [Antibody response to salivary antigen gSG6 was previously shown to be a reliable indicator of human exposure to Afrotropical malaria vectors , 36–38.  gambiae , was fur species  and undeina Faso . Compari species  showed tus group . AnophelSG6 Fig. . On Ambasmission .Anopheles- and malaria-specific antibody responses in Africa [Anti-gSG6 antibody response on Ambae closely followed patterns of decreases in malaria prevalence and community-level antibody responses to all parasite antigens except PvAMA-1. This finding is in agreement with previous reports of malaria incidence and n Africa , 38, 43.n Africa , 45. In An. gambiae gSG6 antigen could be used to measure exposure to An. farauti bites in Vanuatu, the limited conservation between the SG6 protein from these two species  likely result in lower detection sensitivity. Serological analyses using more sensitive biomarker such as cE5 may provide a more accurate measure of exposure to vectors in a population (42). Parasite prevalence in the current study was determined solely by microscopy which likely underestimated the true prevalence. Molecular screening by PCR consistently detects at least twice as many infections as microscopy [P. falciparum and P. vivax infections are common [A number of caveats should be considered in this study. The most obvious one concerns the relatively small number of individuals sampled in each year. As small sample sizes might be sufficient to detect a significant reduction in SCR , but invcroscopy . Recent e common , 48. Thee common . In areae common . Conside19, and AMA-1 from P. falciparum and P. vivax on Ambae Island, Vanuatu between 2003 and 2007. These decreases were matched by a reduction in seroprevalence to the Anopheles-specific salivary gSG6 antigen, suggesting that ITNs were effective in suppressing malaria transmission. This study also demonstrated the utility of recombinant gSG6, originally developed to measure exposure to Afrotropical vectors, in the Pacific where the An. punctulatus group is endemic. Larger follow-up surveys are required to examine more subtle changes in transmission.This study showed the decreases in malaria prevalence and antibody responses to crude SE, MSP-1


Background. Malaria-endemic countries are encouraged to increase, expedite, and standardize care based on parasite diagnosis and treat confirmed malaria using oral artemisinin-based combination therapy (ACT) or rectal artesunate plus referral when patients are unable to take oral medication.Methods. In 172 villages in 3 African countries, trained community health workers (CHWs) assessed and diagnosed children aged between 6 months and 6 years using rapid histidine-rich protein 2 (HRP2)–based diagnostic tests (RDTs). Patients coming for care who could take oral medication were treated with ACTs, and those who could not were treated with rectal artesunate and referred to hospital. The full combined intervention package lasted 12 months. Changes in access and speed of care and clinical course were determined through 1746 random household interviews before and 3199 during the intervention.Results. A total of 15 932 children were assessed: 6394 in Burkina Faso, 2148 in Nigeria, and 7390 in Uganda. Most children assessed (97.3% [15 495/15 932]) were febrile and most febrile cases (82.1% [12 725/15 495]) tested were RDT positive. Almost half of afebrile episodes (47.6% [204/429]) were RDT positive. Children eligible for rectal artesunate contributed 1.1% of episodes. The odds of using CHWs as the first point of care doubled . RDT use changed from 3.2% to 72.9% . The mean duration of uncomplicated episodes reduced from 3.69 ± 2.06 days to 3.47 ± 1.61 days, Degrees of freedom (df) = 2960, Student's t (t) = 3.2 (P = .0014), and mean duration of severe episodes reduced from 4.24 ± 2.26 days to 3.7 ± 1.57 days, df = 749, t = 3.8, P = .0001. There was a reduction in children with danger signs from 24.7% before to 18.1% during the intervention .Conclusions. Provision of diagnosis and treatment via trained CHWs increases access to diagnosis and treatment, shortens clinical episode duration, and reduces the number of severe cases. This approach, recommended by the World Health Organization, improves malaria case management.Clinical Trials Registration. ISRCTN13858170. Plasmodium falciparum malaria now causes 214 million cases and 438 000 deaths, mainly in young African children  $.33), and admitted patients pay for their bed.pm. The policy is that consultations, drugs, and bed costs at public facilities are provided without charge for young children aged <5 years, although caregivers pay for registration cards and injections.In Nigeria, 33 study villages were selected for this study. A similar pattern of care to Burkina Faso is observed—use of traditional healers, traditional birth attendants, CHWs, health centers, maternity wards, and private clinics  as well as shops/patent medicine sellers/drug hawkers where quinine, antibiotics, and antimalarials without prescription are available. In the study sites, there are 8 health centers managed by facility-based CHWs and community health extension workers. The centers are open at all hours if there are admissions; in the absence of admissions they close around 4–6 Uganda has a similar variety of traditional healers, faith homes, drug shops, and private clinics and several grades of health centers. The 84 study villages in Uganda (of which 32 participated in the preintervention study) have 2 grade IV admitting health centers, 1 in each district, with no district hospital. Medical care is provided without charge, but patients pay for hospital registration and medications.Pf], CareStart™ in Burkina Faso, Bioline SD in Nigeria and Uganda) and ACTs (Coartem) supplied in 2 prepacked weight- and age-specific doses were procured by the World Health Organization (WHO) for the study in Burkina Faso and Nigeria and through the Global Fund in Uganda. In Nigeria, the Federal Ministry of Health also provided RDT kits and the Primary Health Centre provided some Coartem. Rectal artesunate was provided for all countries by the WHO TDR  in October 2014. Treatment with rectal artesunate was covered by insurance.Before the intervention, no RDTs, ACTs, or rectal artesunate were routinely available through CHWs in any of the communities. Nationally approved RDTs  or barely utilized (Burkina Faso and Nigeria) prior to the study; thus, the study began by supporting the identification and training of CHWs according to national guidelines in all 3 countries. Communities were visited, the study was explained, and community agreement was obtained to participate. If communities agreed, they were informed that existing CHWs would be selected and trained for the intervention; if not, available community members to be trained as CHWs would be identified using local criteria  before training. In the intervention, each CHW would be responsible for his or her own catchment area.Once CHWs were identified, training was carried out by health staff (Burkina Faso and Uganda) or the investigative team (Nigeria) in sessions that covered theory and practice . TraininTraining was for 5 days in Uganda and 3 days in Nigeria  and 3 days in Burkina Faso. Training was evaluated through pre- and post-tests . A totalAll countries implemented the combined intervention of RDTs, ACTs, and rectal artesunate via trained CHWs from October 2014 to October 2015; Nigeria began earlier with RDTs and ACTs in December 2013.CHWs were not paid in the participating countries in the study. In Burkina Faso, CHWs bought subsidized Coartem and were authorized to retail the same to patients (as per government policy) at 100 XOF (USD $.17) for children up to 36 months and 200 XOF (USD $.33) for children between 37 and 59 months giving them a small profit on each treatment. In Nigeria, CHWs were provided gifts (during festivities) and performance motivation; CHWs’ texts/phone calls to the team regarding specific patients were reimbursed and a stipend was given for team meeting days. Patients were not charged for ACTs in Nigeria and Uganda, and rectal artesunate was provided at no cost to the patient in all countries.In Nigeria, after study initiation, the study team supervised CHWs with visits for review and correction of case report forms (CRFs)/registers, replenishment of drug supplies, and regular monitoring. In Uganda, the 5 nurses, 2 clinical officers, and 3 other facility staff belonging to the 7 facilities in the study areas were assigned supervisory roles and trained to support and supervise CHW performance, manage the supply chain, and support CHWs to manage referrals. Quarterly meetings were organized with transport allowance for CHWs to review progress. Supervision also occurred during a monthly visit posttraining.In each country, meetings were held with local health staff informing them about the study, its purposes and procedures, and their roles in the study, especially for referrals to their facility. In Burkina Faso, 16 nurses and 1 physician (from the only facility) were trained; training included supervision of CHWs. There was refresher training on management of severe patients in the community, facility completion of documentation, tracking of adverse events, commodity and supply accountability, and supervision of CHWs. Field laboratory technicians were retrained in microscopy and storing blood slides for subsequent expert rereading. Medical staff and the inventory controller received stipends. In Nigeria, visits were made to 8 primary health centers in the study area to explain the study to nurses, community health extension workers (CHEWs), junior CHEWs, 1 doctor, and 19 local health staff; all including the malaria focal person for the area were trained. The study covered all training costs including transport. In Uganda, the intervention was organized and implemented by the Ministry of Health. Thus, the procedure for identification, training, supervision, monitoring, and evaluation of CHWs was integrated with routine care procedures for scale-up of integrated Community Management of Malaria (iCCM).Special registers for Burkina Faso and Nigeria and routine iCCM registers in Uganda were utilized to document information on sick children seeking treatment. Every patient assessed was registered and data were obtained on date/time of visit, gender, date of birth, symptoms, known allergies, RDT results, and treatment given. In Burkina Faso and Nigeria, RDT cassettes were collected and reread centrally; In Burkina Faso, blood slides of referred cases were read at the referral hospital and reread centrally . In NigeThe impact of the intervention was evaluated through pre- and post-quantitative household surveys to assess impact (changes in treatment-seeking behavior), qualitative surveys that documented acceptability of the program , and an Except for use of French in Burkina Faso, household interviews were in the local languages , with forms pretested before use. Trained interviewers were fluent in local languages, experienced in data collection, and with a minimum of secondary school certificate in Nigeria and undergraduate degree in Uganda and Burkina Faso. They were trained together, tested in the language of interview and in CRF completion. Ten interviewers were selected in Burkina Faso and Nigeria and 8 in Uganda for the pre-intervention phase and 10 in Burkina Faso, 13 in Nigeria, and 20 in Uganda for the intervention phase.Household surveys were carried out before the intervention and once the intervention had begun. A 2-stage sampling process was used to select households for the survey: First, participating villages were randomly selected, and within villages, households were randomly selected. Households whose caregiver was not present during the illness or who refused to give consent were not interviewed. When a household had >1 sick child within the prior 2 weeks, the first child to be sick was chosen in Burkina Faso, all in Nigeria, but the episode of the youngest child was chosen in Uganda. If the caregiver was absent, the interviewer returned to reattempt the interview; a futile second attempt in Burkina Faso and Uganda and third attempt in Nigeria classified the household as lost for interview.2 test at P < .05.In calculating our sample size for household surveys, we assumed that the average number of children aged <5 years per household was 2, except in Uganda where the average was assumed as 1.4. Requiring a precision of ±5% points for the point estimates of proportions, a minimum sample size would be 384 for the worst-case scenario of 50%. Accounting for a design effect of 2 for clustering on household level would give us a sample size of 2 × 384 = 768 interviews before and during the intervention in each country. We achieved interviews with 1856 caretakers in Burkina Faso , 1560 in Nigeria  and 1529 in Uganda  with an overall total of 4945 households interviewed. Data were entered into EpiData version 3.1. All statistical analyses were performed using Stata software, version 14 (StataCorp). Simple proportions were used for most analyses, and differences between groups were evaluated by χEthical approval for conduct of the study was obtained from the WHO Ethics Review Committee and from national ethical review boards . Informed consent was obtained in accordance with the ethical standards set by the different ethics committees.A total of 15 932 patients consulted trained CHWs during 22 months in Nigeria and 12 months in Burkina Faso and Uganda. About half (53%) of the children seeking care were aged between 6 and 24 months, 71% being <36 months of age  for which patients came for care were febrile, and the majority (82.1% [12 725/15 495]) were diagnosed as parasitologically positive using RDTs (Table Most (98.6% [12 427/12 606]) febrile and afebrile (98.5% [200/203]) uncomplicated malaria cases were treated with an ACT , and almost all febrile  among fewer patients in Uganda to 96.4% (134/139) in a larger series in Burkina Faso. The delay between treatment and arrival at the referral facility ranged from 15 minutes to 31 hours. The median interval was 1 hour in Nigeria (with transport facilitated by the study team), 2.1 hours in Burkina Faso, and 2.5 hours in Uganda. Treatment at the facility for RDT-positive patients was mainly oral antibiotics and antimalarials. CHWs followed up most children at their home within 3 days of treatment; at follow-up, there were no sequelae reported or seen; 2 guardians reported weakness after the episode (1 in Burkina Faso and 1 in Uganda), but the child had recovered by the second follow-up.Seven children treated with rectal artesunate died (5 in Burkina Faso and 2 in Nigeria). Three Burkina Faso fatalities were RDT positive, all treated with rectal artesunate, and went to hospital; the hospital discharge diagnosis was severe malaria, although microscopy at hospital was negative. The remaining 2 Burkina Faso children were RDT negative, 1 with a diagnosis of acute meningitis/bacterial gastroenteritis and severe malnutrition ; the other RDT-negative patient did not arrive at hospital. Two children died in Nigeria: both were RDT positive; 1 had severe anemia, high parasitemia and died 2 days after admission at the hospital while awaiting transfusion; the second child died 30 minutes posttreatment with rectal artesunate, en route to hospital.Community-based diagnosis and treatment was expected to increase use of CHWs, reduce time to malaria diagnosis and treatment, shorten episode duration, and prevent evolution to severe malaria. Household interviews with randomly chosen caretakers of recently ill children were intended to capture changes in treatment-seeking behavior associated with the intervention. Table P < .0001). The use of trained CHWs increased in general (not only as a first resort), from 35.5% (620/1746) before to 50.2% (1606/3199) during the intervention, thus increasing CHW use by 83% .Table P < .0001). ACT treatment through any provider increased from 29.2% (510/1746) to 54.3% (17 37/3199) .Table P = .0014), and mean duration of severe episodes decreased from 4.24 ± 2.26 days to 3.7 ± 1.57 days, df = 749, t = 3.8 (P = .0001).Table P < .0001).We also classified children who had danger signs or not, thus separating the proportion of children with and without severe symptoms before vs during the intervention Table . There wOur results provide strong evidence that providing training, diagnostics, and malaria medicines to CHWs increases coverage, expedites diagnosis and treatment, and shortens the clinical course of both uncomplicated and severe malaria episodes. Severe malaria reflects delays in effective antimalarial treatment. Speed of recovery is important for the child and the family who bear the costs . ReassurWHO recommends that malaria case management be based on parasite diagnosis in all cases . This veP. falciparum malaria infection. Our finding that afebrile children came for care and half were parasite positive is unusual. This finding applies mainly to Burkina Faso and Uganda where malaria prevalence was greatest, suggesting that the results on afebrile malaria may be a feature of high transmission in these 2 countries, as in areas with lower prevalence of parasites, P. falciparum is more likely to result in symptomatic infections. The study did not test for the presence of HbAS hemoglobinopathies that attenuate clinical malaria [For many years, there has been an assumption that fevers are the entry point for diagnosis of malaria and that only febrile children come for care. One speaks of malaria as a febrile illness. Afebrile malaria is rarely recorded. CHWs documented the symptoms of the patients who sought their care, and in about 2.7% afebrile cases, the majority with no other symptoms, they judged that an RDT to confirm or rule out malaria was warranted and found that almost half the afebrile patients harbored a  malaria .The results reflect the pattern of malaria in children presenting to a CHW in the community. Not all who were ill would have sought care from the CHW. Use of RDTs presents the challenge of what to do with children who test negative for malaria, especially when these children are without danger signs. Some studies have shown that the decline in antimalarial treatment accompanying RDT use has had a corresponding effect on antibiotic use . In this