Information on genetic diversity in natural parasite populations constitutes a major impediment to vaccine development efforts and is limited in some endemic settings. The present study characterized diversity by investigating P. falciparum malaria parasitaemic blood screened by light microscopy was depleted of leucocytes using CF11 cellulose columns and the parasite DNA genotyped by nested PCR.Individuals with asymptomatic parasitaemia (AP) or uncomplicated malaria (UM) were enrolled from rural, semi-rural and semi-urban localities at varying altitudes along the slope of mount Cameroon. msp1 allelic fragments (genotypes) respectively. All family specific allelic types  as well as MR were observed in the different locations, with K1 being most abundant. Eighty seven (60 %) of individuals harbored more than one parasite clone, with a significant proportion (p = 0.009) in rural compared to other settings. AP individuals had higher (p = 0.007) K1 allele frequencies but lower (p = 0.003) mean multiplicity of genotypes per infection (2.00 ± 0.98 vs. 2.56 ± 1.17) compared to UM patients.Length polymorphism was assessed in 151 field isolates revealing 64 (5) and 274 (22) distinct recombinant and major P. falciparum in the area and suggests that allele specificity and complexity may be relevant for the progression to symptomatic disease.These results indicate enormous diversity of  Plasmodium falciparum, causes the most severe forms of the disease, is responsible for the high morbidity and mortality, frequent antimalarial drug resistance and aborted vaccines trials . Th. Th20]. Malaria transmission is intense and perennial in the area, with parasitaemia higher in the rainy seasons and at lower altitude . P. falcP. falciparum infection and no signs/symptoms of the disease, asymptomatic parasitaemia (AP), were enrolled through surveys in randomly selected communities as described elsewhere , where n represents the number of samples and pi, the allele frequency at a given locus. Monoclonality was defined as the presence of only one allele of the three major msp1 types in the sample while isolates with two or more genotype were considered as polyclonal infection [The multiplicity of infection (MOI), the number of genotypes per infection was estimated as the average number of PCR fragments per individual, by dividing the total number of eviously . Heteroznfection .χ2 test whereas the differences in group means were assessed using Student’s t - test or analyses of variance (ANOVA). A difference giving a p value ≤ 0.05 was considered statistically significant.All data were entered into Excel and analyzed using SPSS Statistics 20 for windows . The significance of difference in prevalence were explored using the Pearson’s P. falciparum mono-infection were enrolled, mainly with uncomplicated malaria  and asymptomatic parasitaemia . The clinical phenotype of three participants could not be ascertained because their body temperature and/or fever status were not recorded at enrolment. The geographic distribution of the participants included 145 (56 %) mainly from semi-rural areas of mile 16-Bolifamba , mile 14 Dibanda and 15 Buea , Muea  and Mutengene ; 71 (27.4 %) mainly from semi-urban areas of Molyko  and Soppo  and 35 (13.5 %) from rural communities of Tole  and Ombe . The exact residence of 8 (3.1 %) of the participants within the area could not be ascertained. The mean age (± SD) and geometric mean parasitaemia of the participants was 13.83 ± 13.10 years (range: 4 months - 65 years) and 15,715 parasites/μl  respectively. Most of the study participants were from the semi-bantu ethnic group  and had at least 10,000 parasites per microliter of blood . One hundred and sixteen (47.2 %) had anaemia while 30.5 % (40/131) had reportedly taken antimalarial drugs.The characteristics of the study participants are presented on Table P. falciparum field isolates  and 274 major allele family fragments detected respectively, representing an estimated 27 msp1 genotypes. Six (4.0 %) samples  were excluded from the analysis due to negative PCR outcome in all allele families.Length polymorphism was assessed in 151 tes Fig. , with a K1 allelic family was the most polymorphic, with thirteen distinct fragments, representing 10 genotypes while MAD20, MR and RO33 had 8, 5 and 4 different genotypes respectively. All msp1 family specific allelic types i.e. K1, MAD20 and RO33 as well as MR were observed in the different geographical locations  whereas 30 samples contained all three major msp1 allelic types. The overall prevalence of the K1, MAD20 and MR alleles was similar across the different localities whereas the proportion of the RO33 allele (p = 0.007) as well as polyclonality (p = 0.009) was significantly different among the localities. In UM patients, all msp1 allele types were similar across localities while AP individuals in rural settings had the highest proportion of Ro33 (p = 0.003), MR (p = 0.034) and polyclonal infections (p = 0.023)  in asymptomatic parasitaemia (AP) individuals  compared to their uncomplicated malaria (UM) counterparts . However, the proportions of MAD20, RO33, MR and polyclonality were independent of disease phenotype. The variation in the proportion of the recombinant and major allele families as well as polyclonality with age, altitude, antimalarial usage, season and level of malaria parasitaemia in UM and AP individuals is shown on Table MAD20 (p = 0.013) and MR (p = 0.005) alleles was higher in the UM compared to their AP counterparts. Conversely, the proportion of the K1 allele (p = 0.043) was higher in AP children below 5 years compared to their UM counterparts. There was no association between allele proportions with altitude in UM individuals. However, in AP participants, RO33 (p = 0.022) and MR (p = 0.044) allele frequencies were highest at high altitude (Table p = 0.009) was higher in UM compared to AP individuals while at intermediate altitude, the latter had higher K1 (p = 0.009) but lower RO33 (p = 0.043) and MR (p = 0.006) allele frequencies compared to the former. Apart from the higher proportion of the RO33 allele (p = 0.030) in AP individuals during the transition compared to the rainy season, the frequency of alleles was independent of the season of enrolment. During the rainy season, AP individuals had higher K1 (p = 0.017) but lower MR (p = 0.017) allele frequencies compared to their UM counterparts. In UM patients, the proportion of the MAD20 allele (p = 0.041) as well as polyclonal infections (p = 0.041) was higher in non-antimalarial users compared to those who had reportedly taken antimalarial drugs prior to the survey. There was no association between the frequency of alleles or polyclonal infections and the level of malaria parasitaemia  across the different study sites but was higher (p = 0.003) in UM patients (2.56 ± 1.17) compared to their AP (2.00 ± 0.98) counterparts. The variation in multiplicity with age, altitude, locality, season, antimalarial usage and level of parasitaemia in UM and AP individuals is presented in Table p = 0.020) and asymptomatic individuals ≥ 15 years (p = 0.039) had lower mean MOI compared to non-antimalarial users and younger counterparts respectively. Additionally, AP individuals < 5 years (p = 0.009) and ≥ 15 years old (p = 0.007) had lower MOI compared to their UM counterparts. Similarly, MOI was lower in AP individuals at low altitude (p = 0.016), in semi-rural settings (p = 0.011), during the rainy season (p = 0.005) as well as in non-antimalarial users (p = 0.023), moderate parasitaemic (p = 0.007) and high parasitaemic individuals (p = 0.046) compared to their UM counterparts  of the individuals carrying single P. falciparum populations plays a highly important role in the natural acquisition of immunity in malarial infections. Genetic diversity allows parasites to evade natural immune responses, contributing to the failure of anti-malaria parasite control measures and may jeopardize the effectiveness of vaccines [P. falciparum field isolates collected along the slope of mount Cameroon, where malaria is endemic were analyzed. Although the marker has previously been exploited in studying parasites in children from selected foci in the area [P. falciparum genetic diversity in wider population spectrum that includes adults.The genetic structure of vaccines . Therefovaccines . In thisthe area –19 the dthe area . This, tMAD20 and RO33 allele was observed in significant proportion, supporting the notion that sexual intragenic recombination is an important factor in the evolution of genetic diversity [K1 allelic type was found to be the most prevalent, as is the case worldwide. Studies in other areas of the region [K1 allele is more prevalent than MAD20 and RO33. Nevertheless, a recent study in the area [K1 was the least abundant of the three major allele types, consistent with previous reports of MAD20 being the most predominant allele in P. falciparum populations from Myanmar, Thailand, Iran, Pakistan and Colombia [K1 allele prevalence may have been due to the exclusively asymptomatic infections studied, with their inherently low parasite loads [msp1 genetic structure in P. falciparum populations [In addition to all three major msp1 allele families previously reported in the region –19, the iversity . Neverthe region , sub-Sahe region , Nigeriae region  and Conge region  and Indie region  have conColombia . Althougte loads , substanulations . It is aP. falciparum population, with 22 major and 5 MR allelic types (27 genotypes) present at the msp1 locus, the most common not exceeding 30 % of all alleles of a given family. Nevertheless, this is an underestimation of the diversity in the area since 30.5 % of the individuals reportedly received anti-malarial drugs prior to enrolment and clonal disappearance due to treatment activity cannot be ruled out. Although direct comparison could be difficult due to the differences in the volume of blood samples used in each study, the results are consistent with a fast evolution of mutations in an area of high malaria transmission [Genotype analysis showed a very rich polymorphism of the smission . Higher smission , Uganda smission  and Gabosmission  while insmission , Iran [3smission  and Myansmission  parasiteK1 allelic family was the most polymorphic, with thirteen distinct fragments, representing 10 genotypes while MAD20 and RO33 had 8 and 4 different genotypes respectively. These findings are in conformity with previous reports in similar populations in Pakistan [K1, MAD20 and RO33 fragments respectively were observed. Similarly, Bharti et al. [RO33 in India with only 2 fragments detected while 22 K1 and 11 MAD20 were reported. However, in a low transmission area such as Lao PDR, no clonal fluctuation in allelic types are observed in P. falciparum clinical isolates, with monomorphic bands observed for all three major msp1 allele types [The Pakistan  where 12i et al.  confirmele types .K1 allele was higher in AP compared to UM individuals in this study, consistent with the reported association between the K1 allele and asymptomatic parasitaemia in children from mile 16-Bolifamba [K1 allele type may be responsible for the reduced risk of developing symptomatic disease in AP individuals [MAD20 and RO33 were found to be associated with UM corroborating the strong association between the MAD20 allele and symptomatic disease in Ghanaian children [RO33 allele in individuals with UM in mile 16-Bolifamba [MAD20 and RO33 tended to be higher in children below 5 years of age. This suggests that the susceptibility of this vulnerable group of children to UM or symptomatic malaria may accrue to the higher proportion of both allele families. The frequency of the RO33 allele was also significantly higher at high altitude compared to intermediate and low altitude consistent with previous reports of the allele type being the most prevalent at high altitude [MAD20 allele and polyclonal infections were significantly lower in individuals who reportedly took antimalarials compared to those who did not. This could be as a result of selection due to drug pressure on parasite population that is clearing this parasite variant.The proportion of the olifamba  and Nigeolifamba . As suchividuals . Converschildren  as well olifamba . Althougaltitude . There wP. falciparum msp1 is mainly generated by meiotic recombination events involving genetically distinct parasite clones that infect the same mosquito vector. New genotypes and thus increased parasite population diversity do not only accrue to increased number of clones per infected individual but also to the proportion of mixed infections [P. falciparum infections elsewhere in the region [Although it seems likely that nonreciprocal recombination events, such as replication slippage and gene conversion, during the mitotic  replication of the parasite also play a plausible role in creating allele variation, allelic diversity of fections . Multiple region  and in se region . As suchThe overall mean multiplicity of genotypes per infection 2.33) recorded in this study is comparable to previous findings in the area  and to o recordedThis study has a few limitations. First, it is a cross-sectional study, and inherently there are limitations. Though the sample size is reasonable, mount Cameroon is a populous area and there is a definite possibility of sampling error. Furthermore, the inclusion of participants with a minimum of 1000 parasites per microliter only, may have automatically excluded parasite genotypes inherent in individuals with lower parasitaemia.P. falciparum populations in the mount Cameroon area. Multiplicity of infection was high, with most individuals harboring more than one parasite genotypes, reflecting both the high endemicity level and malaria transmission in the area. Allele specificity and complexity are relevant for the progression to symptomatic disease. This data is invaluable in understanding the parasite’s population dynamics which could facilitate the development and testing of a broadly effective malaria vaccine.Intragenic recombination contributes to the high polymorphisms in


Indoor residual spraying (IRS) is considered a valuable transmission control measure against malaria but exact efficacy data are not available for many epidemiological settings. This study was conducted to determine indicators for malaria epidemiology and transmission among school children as baseline assessment before IRS implementation in Ghana.Plasmodium falciparum parasitaemia was detected by light microscopy.A cross-sectional study was conducted in Adansi South District of the Ashanti Region and Wa West District of the Upper West Region of Ghana. Malarial parasitaemia and anaemia were determined in pupils between the ages of 2 and 14 years from Early Childhood Development Centres and primary schools. Out of 1,649 pupils who were enrolled at participating schools, 684 were positive for plasmodia resulting in a baseline parasitaemia prevalence of 41.5%. Parasite rate was similar in the two districts (42.0% in Adansi South and 40.7% in Wa West), but differed across the nine sentinel schools ranging from 21 to 63% (p < 0.001). The mean haemoglobin concentration was 11.3 g/dl [standard deviation (SD) ±2.1]. Pupils who had moderate to mild anaemia (7.0–10.9 g/dl) constituted 41.7% of the study sample.The burden of parasitaemia, malaria and anaemia is a major public health problem among school children in rural Ghana with extensive heterogeneity between schools and warrants further investment in intervention measures. The World Health Organization (WHO) estimates the burden of malaria to be 198 million cases with 584,000 deaths in the year 2013 being a major cause of poverty and low productivity and vice versa  in Adansi South and 40.7% (95% CI 36.9–44.4) in Wa West (Table The mean haemoglobin concentration in the study population was 11.3 g/dl SD ±2.1) (Table .1 (TableBody temperature of the pupils ranged from 36.0 to 39.4°C with a mean of 37.3°C (SD ±0.38). Out of the 1,649 apparently healthy participants attending school, 23 (1.4%) had a temperature ≥38.0°C. Out of these, 11 (0.6%) were positive for parasitaemia and thus fulfilled the case definition of symptomatic malaria and of which nine came from the Adansi South. The cases were equally distributed between the schools.The mean haemoglobin concentration was significantly lower (p < 0.001) in participants with parasitaemia  compared to those without . Corresponding to this, only 47.6% of parasitaemic participants had a normal Hb compared to 61.8%   of the group without parasitaemia  (p < 0.001).Several of the surveyed personal characteristics were associated with a risk of parasitaemia. Table P. falciparum infection is lacking. Nevertheless, microscopy was considered sufficient for the comparison of parasite prevalences before and after IRS intervention given that the same methodology is used.Prevalence of malaria parasitaemia among school children without obvious illness was 42% and 41% in participating schools within Adansi South and Wa West, respectively. While the overall parasite rate in the two districts was similar, there was a strong variation of parasitaemia prevalences across schools. As a tendency, the frequency of parasite carriers without fever was higher in the remote villages  than in the district capital . This finding is in accordance with other studies demonstrating that the most exposed households are those in the more remote parts of a village or district . MicroscPlasmodium infection might have been co-infected with other pathogens causing the fever and resulting in a misclassification as malaria [Notably, 11 of the study participants attending school fulfilled the WHO criteria of malaria. The occurrence of malaria in children at school is in line with findings from other countries in sub-Saharan Africa , 14 with malaria .Similar to the prevalence of parasitaemia, haemoglobin levels and the prevalence of anaemia varied widely between schools. However, there was also significant variation between the two districts because, all cases of anaemia with a haemoglobin <7 g/dl were detected in Adansi South. Interestingly, 9 of the 11 children with fever came from one village possibly indicating a small outbreak of another infection. Furthermore, while our data showed that parasitaemia and anaemia were associated, the different prevalence of anaemia in two districts with similar prevalence of parasitaemia indicated that other co-factors might be involved. Previous studies have shown that causes of paediatric anaemia beyond malaria in Ghana include various nutritional deficiencies, other infections, as well as genetic defects including haemoglobinopathies .Several personal characteristics of the participants were associated with a risk for parasitaemia. As expected, the odds of parasitaemia declined with age. In stable transmission areas such as in Ghana, parasitaemia and malaria symptoms are strongly dependent on the development of semi-immunity after prior exposures .Surprisingly, the data showed that reported use of a bed net in the night before the survey was not associated with any protective effect but, as a tendency, was associated with a slightly higher risk of infection. This finding may be by chance or a result that is confounded by transmission heterogeneity on a microepidemiological scale . Living As anticipated, parasitaemia prevalence among pupils enrolled in the national health insurance scheme was lower than those who were not enrolled. Possibly those with membership in NHIS might have better chance of seeking medical care compared to those without membership. It has been shown that households participating in the NHIS scheme had a higher socio-economic status  and chilThe heterogeneity of parasitaemia and haemoglobin levels was high among schools. Accordingly, a large number of schools must be monitored to provide representative figures for a district. The present data will serve as pre-intervention data for comparison with post-intervention data for the estimation of IRS efficacy. It is recommended to perform regular evaluations over time since the degree of temporal and seasonal fluctuations of malaria and haematological parameters in the study areas have neither been investigated previously nor in the present study. The findings indicate that the burden of malaria and anaemia is a relevant public health problem among school children in Ghana and warrants investment in intervention measures to stem the tide.


Plasmodium falciparum malaria quickly and accurately to reduce malaria related mortality. Existing methods such as microscopy and rapid diagnostic tests (RDTs) have major shortcomings. Here, we describe a new real-time PCR-based diagnostic test device at point-of-care service for resource-limited settings.Sensitive and specific detection of malarial parasites is crucial in controlling the significant malaria burden in the developing world. Also important is being able to identify life threatening ® Malaria, a chip-based microPCR test, was developed by bigtec Labs, Bangalore, India, for differential identification of Plasmodium falciparum and Plasmodium vivax parasites. The Truenat Malaria tests runs on bigtec’s Truelab Uno® microPCR device, a handheld, battery operated, and easy-to-use real-time microPCR device. The performance of Truenat® Malaria was evaluated versus the WHO nested PCR protocol. The Truenat® Malaria was further evaluated in a triple-blinded study design using a sample panel of 281 specimens created from the clinical samples characterized by expert microscopy and a rapid diagnostic test kit by the National Institute of Malaria Research (NIMR). A comparative evaluation was done on the Truelab Uno® and a commercial real-time PCR system.TruenatP. falciparum and P. vivax. The Truenat® Malaria test was found to have sensitivity and specificity of 100% each, compared to the WHO nested PCR protocol based on the evaluation of 100 samples. The sensitivity using expert microscopy as the reference standard was determined to be around 99.3% (95% CI: 95.5–99.9) at the species level. Mixed infections were identified more accurately by Truenat Malaria (32 samples identified as mixed) versus expert microscopy and RDTs which detected 4 and 5 mixed samples, respectively.The limit of detection of the Truenat Malaria assay was found to be <5 parasites/μl for both ® Malaria microPCR test is a valuable diagnostic tool and implementation should be considered not only for malaria diagnosis but also for active surveillance and epidemiological intervention.The Truenat Malaria is a parasitic disease that is endemic in most tropical and sub-tropical ecosystems worldwide with half of the world’s population at risk of contracting malaria , 2. As pPlasmodium and infect many vertebrate hosts, including several species of non-human primates. Five Plasmodium species are parasitic to humans: P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi. Of these, P. vivax and P. falciparum are associated with most malaria morbidity and mortality. P. falciparum is the most dangerous and can cause medically severe form [Malarial parasites belong to the genus ere form . For proere form .Malaria diagnosis is done using tools like microscopy, rapid diagnostic tests (RDTs) involving parasite antigen/enzyme detection and molecular tools (nucleic acid-based). The microscopic examination of a blood smear is considered as “gold standard” for malaria diagnosis in endemic countries. However, the method is laborious and inconsistent , with hiPolymerase Chain Reaction (PCR) is a popular nucleic acid based tool that is used for infectious disease diagnosis. PCR is known to be highly sensitive and specific. In various studies, it has consistently revealed the widespread presence of infections with parasite densities below the threshold level of detection of either microscopy or RDTs ,9,10.The barriers to adoption of PCR include its high cost, and the amount of infrastructure required in terms of equipment and a sophisticated laboratory setup with stable power and refrigerators for reagent storage. Most modern commercial PCR machines are not portable or battery-operated, and so are unsuitable for field or point-of-care applications, especially in areas with low resources. PCR is thus sparingly used in areas of high malaria burden which are largely found in the developing world. Efforts have been made to simplify nucleic acid-based testing by using, for example, isothermal amplification technologies such as LAMP or simple and cheap read-out systems, such as nucleic acid lateral flow immuno-assay (NALFIA) . The NAL® Malaria test  a novel, simple and cost-effective point-of-care technique for differential P. falciparum (PF) and P. vivax (PV) malaria diagnosis using the Truelab Uno® device . The Truelab Uno® device is a portable, light weight, real-time PCR based nucleic acid detection device, operated using a re-chargeable battery power source.Here, in the current study we describe the TruenatThe device weighs 900 grams, and its dimensions are 210 mm X 140 mm X 109 mm (length x breadth x height). It consists of a portable unit housing, an optical detection system, the electronic components controlling different aspects of the unit , a 3.2 i® platform and Truenat® microchips has been reported previously in the detection of Tuberculosis in near-care settings in India [® platform against reference standards used in the field, and found that the platform was suited to use in point-of-care settings in terms of accuracy, ease-of-use, turn-around time and robustness of the device.The use of the Truelab Unoin India , 15. The® platform is described. The performance of the Truenat Malaria test on the Truelab Uno platform compared to expert microscopy and a RDT is also reported.In this study, the development of a novel PCR-based malaria test that runs on the Truelab Unowww.ncbi.nlm.nih.gov/tools/primer-blast/) ClustalW software  was used for in silico sequence alignment analysis. The assay was first standardized and characterized on a conventional real-time PCR device  and then ported to the Truelab Uno® system where the PCR reaction happens on a microchip. The final result after the Truenat® Malaria test is run on the Truelab Uno® system is provided as genome equivalents per μl for positive samples.The proprietary Truenat Malaria assay was developed and standardized by bigtec. The target genes for both PF and PV are single copy genes. The assay is based on Taqman chemistry. The probes and primers were designed and characterized using tools like Primer Express (Applied Biosystems), Primer3 (simgene.com/Primer3) and NCBI Primer Blast  [This cross-sectional paired study design was chosen to determine the performance of the Truelab Uno, India) . A team A total of 281 patients were recruited for this study. Study samples were collected from Wenlock Government District Hospital, Mangalore, Karnataka from August 2009 to October 2010. Samples were also collected from two malaria endemic villages in Koraput district, Odisha from July to November 2009. Patients of either sex, from 3 to 62 years of age, presenting with fever or recent history of fever, were chosen for participation in the study. Finger pricked whole blood samples (approximately 50μl) were collected in EDTA coated vials and stored at -20°C during this period. The DNA from blood samples was extracted using QIAamp DNA blood Mini kit.Finger pricked thick and thin whole blood smears were prepared on the same slide. The thin smears were fixed with Methanol and the thick smears were dehaemoglobinized with distilled water. These smears were stained with 10% Giemsa. Subsequently, these stained smears were examined on double blind mode independently by two expert microscopists. Any discordant result was resolved by a Senior Scientist (SKG). Final readings were calculated on the basis of average of the two microscopists. A smear was declared negative after thorough scanning of 100 micro-fields on thick films. Quality control was performed by randomly choosing 10% of the smears for analysis by the Senior Scientist (SKG). Parasitaemia was calculated against 200 white blood cells on thick film assuming 8000 WBCs/μl blood for a single patient .Falcivax™ is a rapid, qualitative, two site sandwich immunoassay utilizing whole blood for the detection of P. falciparum specific histidine rich protein-2 (PfHRP-2) and P. vivax specific pLDH. The test can also be used for specific detection and differentiation of P. vivax and P. falciparum malaria [ malaria . Test waBlood samples were collected from suspected malaria patients residing in endemic areas in EDTA vials using finger prick method. After completing the microscopic examination and RDT, the remaining blood was used for PCR analysis. DNA was isolated from the remaining blood samples using QIAamp DNA blood mini kit according to the instructions as per the handbook of QIAamp DNA blood Mini kit. For amplicon purification from agarose gels, QIAquick gel extraction kit was used as per manufacturer’s instructions. Proprietary malaria-specific primers and probes were synthesized by Sigma. The master-mixes used for PCR are proprietary components of the Truenat Malaria kit.Real-time PCR on ABI 7500: 4 μl of extracted DNA was mixed with 6 μl of the Truenat malaria mastermix and real-time PCR was performed on ABI 7500  under the following cycling conditions:Step 1:95°C for 60 secStep 2:95°C for 5 secStep 3:60°C for 34 secSteps 2 & 3 were repeated for 40 cycles™ microPCR: 5μl of extracted DNA was added to the Truenat malaria microchip and the real-time PCR was done using a pre-programmed profile on the Truelab Uno™ microPCR. Results were observed on the screen and compared to the results obtained on the ABI 7500 using the same mastermix.Real-time PCR on Truelab UnoP. falciparum and P. vivax clinical samples was used as starting material to generate the relative standard curves for qPCR. 100 μl of whole blood was processed using the QIAamp DNA blood mini kit. 25 μl of the extracted DNA was subjected to PCR along with the respective primers using a conventional PCR machine . After PCR the amplified samples were run on an agarose gel and stained with ethidium bromide. The amplicon band was then excised from the gel and purified using a Qiaquick gel extraction kit. The absorbance (2μl of DNA) was estimated at 260nm using a NanoDrop 2000  following the manufacturer’s instructions. The copy number of the pure amplicon was calculated as given below. Serial dilutions of a known amount of amplicon DNA were used to produce a standard curve in the form y = mx + c, where m represents the gradient and c represents the y intercept. The PCR was performed in triplicates on the Truenat Malaria chips run on the Truelab Uno system and the average Ct was used for plotting the standard curve. The standard curves for the PF and PV were flashed onto the Truenat® Malaria chips in order to convert the Ct obtained to genome equivalents/μl.Genomic DNA from Nanomoles of amplicon were calculated using the following equation:Extinction coefficient of the DNA was calculated from individual base coefficient by summing up.Copy number was calculated using the formula:P. falciparum DNA nucleic acid amplification technology (NAT) assays, obtained from the National Institute for Biological Standards and Control  was used as the calibration reference reagent for the P. falciparum assay. The standard consists of a freeze-dried whole blood preparation collected from a patient by exchange transfusion. As per instructions in the product data sheet, this lyophilized material was suspended in 500 μl of sterile, nuclease-free water to a final concentration of 109 IU/ml. As per a previous study [P. falciparum reference material detectable by the Truenat® Malaria assay.The WHO international standard for us study , this coP. vivax, in absence of a reference material, genomic DNA was used. The extraction and quantitation of the genomic DNA are described in Section 2.9. Serial dilutions of the DNA were subjected to PCR in replicates to estimate the lowest number of P. vivax genome equivalents that were detectable by the Truenat® Malaria assay.For estimating the lower limit of detection of ™ microPCR with expert microscopy and rapid diagnostic test (RDT), which are the standard tests for malaria detection, a study was done on a sample panel consisting of 281 clinical samples including 6 follow-up samples  collected from two different malaria endemic areas. The samples were subjected to malaria detection by expert microscopy, RDT and Truenat Malaria test on Truelab Uno™ microPCR by different technicians who did not know the identity of the samples prior to testing. The data was compiled by NIMR and the results were unblinded by an expert committee. The percentage sensitivity of the Truenat® Malaria and the RDT tests using microscopic examination of the blood smears as the gold standard was calculated as follows: true positives/ × 100. In addition, 95% Confidence Intervals (95% CI) were calculated using an online calculator http://vassarstats.net/clin1.html.In order to determine the specificity and sensitivity of Truelab UnoThe study was approved by Institutional Ethics Committee of Kasturba Medical College, Mangalore—a constituent college of Manipal University, Karnataka, India. The institutional Review Board approved the protocol, which was implemented in accordance with the Helsinki Declaration and regulatory guidelines of ICMR, Government of India. The study protocol was also approved by the Scientific Advisory Committee of National Institute of Malaria Research (ICMR), New Delhi. Finger pricked blood samples were collected from all participants after obtaining written informed consent by themselves or their parents or guardians. Written assent was also taken from the guardians of children who were able to understand the study process, when required. Truelab Uno results were not used for clinical decision making and the patient samples used were anonymous with no identifiers to patient identity.P. falciparum and P. vivax detection and quantificationBased on the standard curves generated using genomic DNA, the Truenat Malaria assay was found to be linear over 6 orders of magnitude for P. falciparum DNA nucleic acid amplification technology (NAT) assays, obtained from NIBSC, the lower limit of detection for P. falciparum is estimated to be approximately 4.7 parasites/μl. With the use of the DNA from a P. vivax positive clinical sample, the lower limit of detection of the P. vivax assay was found to be 10 genome equivalents per PCR reaction. Based on the relation between genome copy number and parasitic load (parasites/μl) established in a previous study [® Malaria is estimated to 1.3 parasites/μl for P. vivax.Based on the use of the WHO international standard for us study , the lim® results were categorized as positive or negative. The parasitaemia information obtained (from coversion of Ct into parasites/μl based on the standard curve) was ignored for this analysis. The Truenat Malaria test identified all 50 P. falciparum positive samples correctly and was negative for P. vivax in all of these samples. Similarly, the Truenat Malaria test identified all 50 P. vivax positive samples and did not detect any of these as P. falciparum positive. This gave an analytical sensitivity and specificity of 100% each at the species level . So for comparison, Truenatl Tables .® to verify whether the result obtained on the Truelab® was the same as that obtained using a commercial thermal cycling unit. It was found that the results obtained from the Truelab Uno® microPCR were the same as that obtained using the commercial ABI 7500 machine for all 281 samples, with no device related artifacts introduced by the thermal cycling process on the Truelab Uno®. The overall time taken to complete the PCR starting from DNA isolation was approximately about 45 mins with the Truelab Uno™ microPCR device while it was around 1hr and 30 mins with the commercial ABI 7500 machine.The PCR was performed in parallel on the ABI 7500 and the Truelab UnoP. falciparum and P. vivax by expert microscopy. Of these 141 negative samples, 117 were negative for both P. falciparum and P. vivax by Truenat Malaria, and 129 were negative for P. falciparum and P. vivax by RDT  or P. vivax positive (71 of 140), or mixed infection with both P. falciparum and P. vivax positive (4 of 140). The Truenat Malaria test identified 64 of 65 P. falciparum positive samples, all 71 P. vivax positives and all 4 mixed infection samples correctly giving a sensitivity of 99.3% at the species level . Only 4 of 32 were picked as mixed infection by expert microscopy and 5 were picked as mixed infection by RDT. Of the 32 samples, 19 were detected as P. falciparum positive only by both expert microscopy and RDT and 4 as P. vivax positive only by both expert microscopy and RDT. In 19 samples, the P. vivax parasitic load detected by Truenat Malaria was greater than 3000 genome equivalents/μl. Among the 4 samples, P. falciparum parasitic load was less than 8000 genome equivalents/μl in all, and as low as 460 genome equivalents/μl in one of the samples. All the raw data for the 281 samples has been provided as Supplementary file . The sample processing is done semi-automatically with the user following pipetting instructions as they appear sequentially on the device’s alpha-numeric LCD screen  was not performed. This was because there was not enough blood remaining after performing the microscopic examination and RDT to extract enough DNA to run the Truenat® Malaria test on a commercial thermal cycler and Truelab Uno® and then also performing the nested PCR test. True negative status of the 281 samples could not be established in the absence of nested PCR results. Since expert microscopy failed to detect numerous samples that were positive by Truenat Malaria owing to its higher limit of detection, specificity of the Truenat® Malaria could not be determined using expert microscopy results as a reference for the 281 sample panel.For the clinical sample panel of 281 samples tested using Truenat® platform was not complete when this study was initiated, all sample processing were done using commercially available kits. Multicentric studies need to be initiated to test the performance of the Trueprep MAG® device in the diagnosis of malaria in different field settings.As the development and validation of the Trueprep MAG® microPCR platform was found to be highly useful in rapid, sensitive diagnosis of malaria infections. It was also found to be efficient in detecting cases of mixed malarial infections. Its use could be extended to detection of malaria in active surveillance programs.The Truenat malaria test on Truelab UnoS1 Table(XLS)Click here for additional data file.


Plasmodium falciparum malaria in nonimmune adults and a major contributor to their mortality. In children, renal failure, though not very common, has become a rising issue leading to death. This study aims at determining the incidence of renal complication in malaria cases reported in children of Odisha. 108 cases of malaria who were admitted to Department of Paediatrics, SCB Medical College and Hospital and Sardar Vallab Bhai Patel Post Graduate Institute of Paediatrics, Cuttack, Odisha, India during the period from July 2006 to November 2008 were included in the prospective study. Extensive investigations were carried out to check for renal involvement in these cases. 50.9% of cases showed some form of renal involvement, most of which were recorded in age group of 5–10 years. Overall, males had a higher incidence than females. 62.7% of total cases infected with P. falciparum showed renal involvement though mixed infections with both P. falciparum and P. vivax had 100% renal involvement.About 500 million people suffer from malaria leading to death in 2-3 million cases every year, of which about 1 million are children. Horstman et al., 1985, and Weber et al., 1991, demonstrated an acute renal failure as a well-described complication of  Plasmodium falciparum [P. falciparum malaria in non-immune adults and a major contributor to their mortality. In contrast major manifestations of severe falciparum malaria in children are cerebral malaria, severe anemia, metabolic acidosis [Malaria is now a leading public health problem in developing world including India. Almost all complications and death from malaria are caused by lciparum . Recentllciparum . Some stlciparum  demonstracidosis , but renacidosis .falciparum malaria. The overall incidence of ARF reported by various Indian authors ranges between 4 and 17.2% [falciparum malaria is 7.7% in under-5 children and 18.4% in 5–14-year children [The clinical spectrum of renal involvement in malaria varies widely from urinary sediment abnormalities, mild proteinuria, electrolyte changes to acute renal failure (ARF) with metabolic acidosis as well as nephritic syndrome. Among renal manifestations, ARF is the commonest manifestation of severe nd 17.2% . StudiesThe clinical syndrome of quartan malarial nephropathy  is nonspP. falciparum nephropathy can manifest as acute tubular necrosis, acute interstitial nephritis, and glomerulonephritis. The incidence of acute tubular necrosis is estimated at between 1 and 4% of all cases of falciparum malaria, but it may reach 60% in cases clinically categorized as being malignant. Prognosis depends on the severity of the condition, associated extrarenal complication, response to anti-parasitic treatment and availability of dialysis. The reported mortality ranges from 15 to 30% [5 to 30% –11.P. falciparum acute tubular necrosis and acute glomerular nephritis [falciparum malaria at any age, although children remain the main population at risk [falciparum glomerulopathy is reversible within 2–6 weeks upon the eradication of the infection.Although isolated interstitial nephritis has not been reported in humans, interstitial inflammation is a common histopathological finding in ephritis , 13. Glo at risk –16. It i at risk  and acut at risk , 19 syndResurgence of malaria in Odisha provided an opportunity to study the different renal manifestation of malaria in children and its correlation with severity of infection and clinical presentation. This study aims at determining the incidence and renal involvement in different types of malaria.The study was conducted in the Department of Paediatrics, SCB Medical College and Hospital and Sardar Vallab Bhai Patel Post Graduate Institute of Paediatrics, Cuttack, Odisha, India from July 2006 to November 2008. All cases admitted to the paediatric ward with smear-positive or ICT-positive malaria were taken up as subjects for the prospective study. Patients with preexisting renal disease were excluded from the study. Cases with clinical features of malaria with asexual form of plasmodium in the peripheral smear seen by thick and thin smear were taken into consideration. It included 108 cases of malaria, diagnosed either by smear examination or rapid diagnostic test. Other investigations like hematological tests, biochemical tests, and urine examination were done. Smearing was done under aseptic conditions and stained to examine under oil immersion objective of light microscope. The nucleus of the malaria parasite looks pink and cytoplasm blue in color. Thick blood film increases sensitivity of the parasite detection because of more parasite concentration even though the morphology of parasite is distorted. One microscope field is equivalent to 50 microscope fields of thin film. Hence it is more useful for mass surveys and a guide to assess the antimalarial response.P. falciparum was identified by the following characteristic features:two nuclei on same side of the ring/opposite side of ring,multiple infection of RBC, accole form of trophozoite, the size of the infected RBC does not alter,sickle-shaped gametocytes which are one and half size of RBC.P. vivax was identified by the following characteristic features:one parasite in a RBC,cytoplasm is thick with single nucleus, parasitized RBC is larger than normal, accole is not seen.Detailed evaluation was done with special reference to urine output and presence of edema. All cases were subjected to investigations such as serum urea, creatinine, electrolyte, and urine analysis to check for renal involvement. Deviations from the normal range of variables are extrapolated as markers of renal pathology. Blood urea nitrogen and creatinine ratio was calculated for each patient from the tabulated data and it was inferred whether the renal involvement was due to prerenal causes or of renal etiology. If urea creatinine ratio was >20 mg/dL, then it was considered as pre-renal cause and urea creatinine <20 mg/dL as renal causes.If any one of these above criteria is fulfilled, then it is considered as renal involvement present.Out of the 108 cases studied, there were 60 (55.5%) male and 48 (44.5%) female cases with a male-to-female ratio of 1.25 : 1. This coincides with the fact that male children are usually less covered and exposed more to mosquito bites. A maximum number of cases were detected in the age group of 5–10 years (62%) followed by 10–14 years (27%), and 0–5 years (11%). As seen from falciparum malaria has renal involvement as compared to only 28.6% of vivax cases. All mixed infection cases have renal involvement. 62.7% of patient with P. malaria infection. At the other extreme is a severe disease dominated by the hemodynamic consequences of massive red-cell parasitization, eventually leading to acute tubular necrosis. This is mostly seen with P. falciparum infection. In between lay two syndromes, namely, acute interstitial nephritis and acute proliferative glomerulonephritis.Malaria reemerged once again as a major public health problem of India in the late 1970s after a significant decline in the 60's, affecting 2–2.5 million cases usually. Thus malaria is a parasitic diseases of great epidemiological importance, and malarial renal involvement is emerging as an important problem in tropical countries. Renal involvement in malaria displays the full spectrum of interaction between red-cell abnormalities and TH1 and TH2 activation. At one extreme is a slowly progressive immune-complex mediated glomerulopathy that supervenes in a setting of TH2 predominance, classically associated with In the study 50.9% have some form of renal involvement. Similar results were seen in different studies, 30–70% , 46.9% , 53–46% In another study, maximum cases observed in age group 0–5 years . But in  In this study 62.7% of patient cases have some form of renal involvement. Similarly other studies have given comparable results. Few studies give 58.2% of renal involvement in falciparum malaria, 23% in vivax malaria . The perIt is concluded that male children of 5–10 years are more vulnerable for malaria with renal involvement more so if mixed infection is present. Early and prompt diagnosis along with aggressive therapy can prevent progression to complicated malaria while we are waiting to combat other factors like vector control, administrative shortfalls, financial stringency, and so forth. A new molecular marker has to be searched for, and that may give early clue to renal involvement before biochemical abnormality of renal involvement is detected.


Sir, We read with interest the detailed review by Plasmodium falciparum malaria, as shown in an autopsy study (The authors omitted to discuss pressure autoregulation, and vascular response to carbon dioxide and oxygen in the brain and retina. Cerebral autoregulation seems to be impaired .Retinopathy is observed in a proportion of Bangladeshi adults with cerebral malaria (


Plasmodium falciparum malaria is estimated to affect young children in sub-Sahara Africa, where it remains a common cause of hospital admission and inpatient mortality. Few children will ever be managed on high dependency or intensive care units and, therefore, rely on simple supportive treatments and parenteral anti-malarials. There has been some progress on defining best practice for antimalarial treatment with the publication of the AQUAMAT trial in 2010, involving 5,425 children at 11 centres across 9 African countries, showing that in artesunate-treated children, the relative risk of death was 22.5%  8.1 to 36.9) lower than in those receiving quinine. Human trials of supportive therapies carried out on the basis of pathophysiology studies, have so far made little progress on reducing mortality; despite appearing to reduce morbidity endpoints, more often than not they have led to an excess of adverse outcomes. This review highlights the spectrum of complications in African children with severe malaria, the therapeutic challenges of managing these in resource-poor settings and examines in-depth the results from clinical trials with a view to identifying the treatment priorities and a future research agenda.Over 90% of the world’s severe and fatal  Plasmodium falciparum malaria each year [P. falciparum malaria falls on sub-Saharan Africa (sSA), where children under five years old are disproportionately affected by this parasite  and other vector control measures. Nevertheless, in 2010 it was estimated that there were 500 million episodes of ach year . The hean in sSA ,5.FigureSevere malaria is a complex multisystem disorder with a clinical presentation that has many similarities to severe bacterial sepsis, despite distinctive differences in pathogenesis. Children typically present when they are critically ill with life-threatening complications yet, in most African hospitals, few will ever be managed on an intensive care unit or in a high dependency facility. The high, and frequently seasonal, burden of cases of severe malaria presents a unique challenge to health services and clinicians working in resource-limited hospitals. This review intends to provide an overview of the spectrum and complications of severe malaria, highlighting treatment priorities for children managed within low-resource settings in Africa and re-examining what has been learnt from clinical trials to outline a future research agenda. In order to contextualise the current and future relevance of this review and the continued necessity to prioritise research funding for this condition, I will briefly summarise the current status of malaria eradication efforts, aimed at reducing the disease burden and mortality, and accordingly, if successful and sustained, negating the necessity for future investment in this clinical disease.Lancet series [The global strategy towards gradual malaria eradication which has been developed and ratified into policy was reviewed in 2010 in a four-part t series -9. It fot series . Howevert series , suggestt series , an incrt series ,16 or a t series . The dowP. falciparum malaria, were encouraging. However, longer term follow-up indicated these responses were imperfect and short-lived. In the 18 months following vaccination with RTS,S, vaccine efficacy (VE) against clinical malaria in children was 46%  42% to 50%) but only 27% (95% CI 20% to 32%) in infants vaccinated between 6- and 12-weeks old [With regard to vaccine development, immunological and clinical data from large Phase III multicentre vaccination trials of the most promising candidate on the market to date , a vaccine which is directed against the pre-erythrocytic stage of eeks old . VE wane% 95% CI % to 32% P. falciparum malaria infection, is distinguishing those who are at greatest risk of poor outcome, using largely clinical criteria, in order to target parenteral antimalarials and supportive therapies [Formerly, severe malaria in African children was considered to comprise two distinct clinical syndromes: cerebral malaria and severe malarial anaemia. This paradigm was supported by clinical, molecular, immunological and genetic studies and, indeed, substantially influenced their experimental design. As a consequence, it was held that most malaria deaths were attributable to cerebral malaria and, thus, were primarily neurological in origin, with a smaller number resulting from severe malarial anaemia, which could be mitigated by an immediate blood transfusion. Over the last three decades substantial research funding has enabled much clearer and detailed clinical phenotyping of severe malaria in African children, its pathophysiology and complications. What has been determined is that severe malaria encompasses a complex syndrome affecting many organs resulting in biochemical and haematological derangements which have many features in common with the pathophysiological derangements complicating children with severe sepsis. Moreover, in-hospital deaths, most occurring within 24 hours of admission, appear to be a consequence of a wide spectrum of pathophysiological determinants. At the clinical level, a key challenge for health services in Africa, owing to the large burden of paediatric admissions with herapies . This isP. falciparum malaria could either have severe malaria or very severe pneumonia, an issue that has been covered in a number of publications [P. falciparum malaria in Kenya established that two clinical features, impaired consciousness (defined as coma or prostration) and respiratory distress , identified 84% of fatal cases. Prostration (the inability to sit upright in children over eight months or breast feed), any respiratory distress, hypoglycemia, jaundice, or any combination of these, predicted 92% of fatal cases. Relevant to the traditional severe malaria paradigm was that severe malarial anaemia (defined as a haemoglobin (Hb) <5 g/dl) in the absence of these two clinical signs had a very low mortality (1.3%) [Differentiating ‘true cases’ of severe malaria from other acute infections remains both a clinical and epidemiological challenge, as most symptoms of malaria are indistinguishable from other major causes of morbidity, in malaria-endemic sSA where parasite carriage is the norm rather than the exception. For example, a child with severe respiratory distress and ications -26. Detey (1.3%) . Similary (1.3%) . Using dy (1.3%) . Probably (1.3%) . AQUAMATy (1.3%) . These wy (1.3%) . The malThe latest WHO guidelines , informeNeurological involvement is common in African children. Typically, children with cerebral malaria present with a history of a febrile illness lasting one to three days with convulsions, impaired consciousness, with or without brain-stem signs. This is strictly defined as an unrousable coma that persists for more than one hour following a seizure (irrespective of anticonvulsant medication) since many children with malaria regain full consciousness after a brief convulsion. The most widely used paediatric classification of impaired consciousness in sSA is the Blantyre Coma Scale (BCS), a practical tool developed for children who are too young to speak ,35. ComaNeuroimaging data describing the changes associated with cerebral malaria in African children remains very limited. The only prospective study using magnetic resonance imaging (MRI) involved 120 Malawian children with cerebral malaria  . It idenP. falciparum malaria, manifesting as either simple tonic-clonic or partial convulsive episodes [P. falciparum malaria parasitaemia [Seizures are the most common neurological complication of acute episodes  to cliniepisodes , which mepisodes . Overallsitaemia . Whilst sitaemia .In addition to seizures, abnormal motor posturing (AMP) is also a common complication of children with cerebral malaria, manifesting as generalised extension of the trunk, increased muscular tone and posturing in the upper and lower limbs and clinically characterised as de-corticate, de-cerebrate or opisthotonic posturing. Whilst the aetiology and pathogenesis remains poorly understood, Idro and colleagues attempted to define the burden and the prognosis of each type of posturing. In 417 Kenyan children with cerebral malaria, 163 (39.1%) had posturing during hospital stay, with 50% developing AMP after admission , indicatOutcome for cerebral malaria remains poor with high in-hospital mortality and neurological sequalae in survivors. In the AQUAMAT trial the subgroup with the greatest mortality were children with coma, with an overall mortality of 359/1,825; 19.6% . OutcomeP. falciparum malaria in African children, particularly young infants in high transmission settings. It is caused by haemolysis of both infected and uninfected erythrocytes and exacerbated by impaired erythropoiesis [Severe anaemia in children in sSA remains a leading cause of hospital admission  and a maopoiesis . Within opoiesis .In terms of prognosis, since the prevalence of pre-morbid chronic mild to moderate anaemia is very common in these populations, presentation in the absence of any other complications has a lower MAF  and carrImproved diagnostic capabilities have led to a better understanding of the importance of metabolic acidosis as a common complication of severe malaria, despite its clinical correlates (deep ‘Kussmaul’ breathing) being recognised for decades ,19. HistDespite being a common complication of severe malaria, the aetiology of malarial acidosis is not well understood. At a clinical level metabolic acidosis is more frequent in children with severe anaemia , hypovolP. falciparum malaria, especially in children and pregnant women. Ten percent of children enrolled in the AQUAMAT trial had hypoglycaemia (glucose <3 mmol/L) at the point of admission [Hypoglycaemia is an important complication of severe dmission  whereas dmission -74. Hypodmission ,53,75, sdmission ,71,76,77dmission ,73. If ndmission ,54,75. Tdmission . Hyperindmission ,80. Datadmission .P. falciparum malaria [In clinical practice there are several definitions of hypoglycaemia, each targeting a different blood glucose threshold. In adults and children hypoglycaemia defined as a blood glucose of <2.2 mmol/L has been shown to be associated with a poor outcome  and, hen malaria . Include malaria .As in sepsis, dysregulation of the cortisol, insulin, mitochondrial, or other metabolic pathways have been implicated in the aetiology of hypoglycaemia and deserve further investigation. The evidence supporting the current threshold suggests that intervention at a higher cutoff maybe warranted but this needs to be tested in an adequately powered clinical trial directly investigating the optimum threshold for treating hypoglycaemia and the best mode of treatment (dextrose loading or infusion).2 < 90%), hypoglycaemia (<2.5 mmol/L) and creatinine >80 mmol/L  P = 0.0002). In the AQUAMAT trial 1,009/4,148 (24%) had a BUN >20 mg/dL [Renal involvement in severe malaria is common in adult non-immune case series and semi-immune cases in south- and southeast Asia (around 25% to 50% across various series). Patients may progress to acute kidney injury with anuric or oliguic renal failure . However20 mg/dL  and this20 mg/dL . The mor20 mg/dL   and direct tubular or glomerular injury. Pathological studies in adults with severe malaria have shown sequestration of parasitised red cells in glomerular and tubulo-interstitial vessels and acute tubular damage due to accumulation of monocytes in glomerular capillaries. The level of sequestration in the kidney was greater in those who developed renal failure than in fatal cases without renal failure . No equiP. falciparum malaria infection, aetiological factors included quinine treatment (56%) and G6PD deficiency (54%) [P. falciparum malaria transmission , and Uganda) [Haemoglobinuria, or blackwater fever (BWF), a syndrome comprising acute intravascular haemolysis, fever and the passage of dark- or red-coloured urine, in patients with current or recent malaria infection, is recognised as a potential complication of malaria. Classically, it was noted to be a consequence of long term use of quinine in non-immune expatriates -94. In Acy (54%) . In Afri Uganda) -100. In  Uganda) . In Nige Uganda) .There is, however, renewed interest in this topic since there have been a number of case series reported from Europe of delayed haemolytic anaemia following treatment of severe malaria with injectable artesunate ,103. AllP. falciparum malaria (describing IBI) across sSA examined the evidence supporting a biological or epidemiological relationship [Escherichia coli and other enteric gram-negative organisms. There was also some evidence indicating greater risk of NTS IBI in children with malarial anemia [Bacteraemia is an important complication of severe malaria in African children but is often under recognised, largely a result of limited and poorly maintained microbiology services across sSA. Nevertheless, data have slowly emerged, mainly from hospitals linked to research programmes, indicating that there may be a biological link between malaria and susceptibility to invasive bacterial infection (IBI). A study in Teule, Tanzania, reported that up to a third of paediatric severe malaria deaths were attributed to bacteraemia . Anothertionship . In the l anemia .P. falciparum malaria infection plus one or more of: prostration, respiratory distress (chest indrawing or deep breathing), severe anaemia (Hb <5 g/dL), or HIV infection. A pathogen was isolated from aerobic blood cultures in approximately 20% of children (1-week to 12-years old) meeting these criteria. The ‘Teule criteria' are yet to be tested in prospective intervention trials, which could help with future policy of directed  or short-course empiric therapy with early termination guided by culture results to prevent development of resistance, which is commonly practiced in high-income countries [In order to reduce IBI-associated malaria mortality, whilst minimising the risks of excess antibiotic prescribing, further research is needed to establish children at greatest risk of bacteraemia, to inform a policy for targeted antibiotic therapy. For example, Nadjm and colleagues proposed simple clinical criteria  which idountries .P = 0.0002).Quinine has been the mainstay of treatment for African children with severe malaria for decades; it remains effective and little drug resistance has been reported from the continent. The arrival of the artemesinin compounds led to a series of small clinical trials to test their safety and efficacy. The first comparative clinical trials investigated artemether , which because of its poor water solubility can only be injected by the intramuscular route. In clinical trials it proved to be safe but when the data on efficacy were collated in a meta-analysis  it did not lead to an improved overall survival, specifically in African children. This was followed by trials investigating another artemisinin formulation, artesunate, which in contrast to parenteral artemether, is water-soluble and can be given intravenously and is instantly bioavailable . Even inP. falciparum malaria infection. The primary endpoint was in-hospital mortality. By intention to treat, 297 children (10.9%) receiving quinine treatment died compared to 230 (8.5%) children who received artesunate. Overall, this translated to a relative reduction in mortality of 22.5% (95% CI 8.1 to 36.9) in children in the artesunate arm (P = 0.002) [P = 0.02); but there were no other differences between the arms in number of other complications, additional treatments prescribed, time to recovery or numbers with neurological sequelae – which included 61 persistent mild, moderate or severe such sequelae among survivors [A further trial in African children was justified since expert review at that time considered that the issue of parasite drug resistance to quinine was not an issue (as it was in South East Asia), that quinine remained the optimal antimalarial and owing to the rapid tempo of severe disease in African children with severe malaria compared with adults in South East Asia there were questions about the generalisabilty of the SEAQUAMAT trial. In severe malaria in children most deaths occur within the first 24 hours whereas survival benefit for many patients in the SEAQUAMAT trial occurred after this time point. These issues highlight the importance of equipoise as a strong scientific justification and a prerequisite to conduct further clinical trials with meaningful clinically relevant endpoints . The AQUAMAT trial enrolled children at the point of hospital admission. The criteria for enrolment were broad and pragmatic with a view to future generalisability of the trial results, principally if the clinician considered the child required parenteral antimalarial medication they were eligible for inclusion in the trial. Diagnosis was reinforced by quality controlled laboratory evidence of urvivors .The outcome of severe malaria is largely determined by the complications and number of vital organ dysfunction and quality of care available. For most African children this will not include an admission to an intensive care or high-dependency care unit, instead management will be more usually on a busy paediatric ward. It is important to emphasize that the most important factor for management is the implementation of specific antimalarial drug treatment and that this is given as early as possible to reduce the risk of adverse outcome. As is the case in management of severe sepsis, supportive therapies aimed at correcting deranged physiology are also fundamental to improving outcome; however, of the adjunctive agents tested in clinical trials in both adults and children all have failed to show benefit, with the exception of renal replacement therapy in adults . With reIn general, many of these trials were single-centre phase I or II studies with low sample sizes, often constrained by funding and slow or truncated trial recruitment. A number of trials were terminated early due to political instability, meeting pre-defined safety stopping rule or due to evidence of adverse outcome in the interventional arm. Many trials had primary endpoints targeting clinical surrogates for prognosis  and so, as is usual in Phase II trials were not powered to inform meaningful endpoints such as mortality, but are important steps in the justification for further testing in larger trials. Methodological issues were noted in some trials, for example, in some the primary endpoint was not pre-specified, methods of randomisation were not reported and some trials appeared to recruit patient populations with substantially lower overall lower mortality than would be expected, thus reducing the external validity of the results. How we interpret these trials is possibly helped by one overarching emerging theme: that most did not result in a positive outcome for any of the patient-centred endpoints in the interventional arms . Ordinarily, the early stopping of trials, especially when interim analysis suggests large treatment effects, has been criticised as, more often than not, interim results on small numbers tend to overestimate treatment effects  which arIn order to reduce or prevent cerebral oedema, two separate trials investigated whether the addition of dexamethasone improved the outcome of cerebral malaria in adults and children ,115. TheThe pathogenesis of severe malaria involves several different processes, including enhanced production of the cytokines including tumour necrosis factor (TNF), sequestration of parasite red cells to the endothelium and decreased erythrocyte deformability. Since pentoxfylline (Pt) has beneficial effects on many of these processes, in particular increasing red cell deformability , it was postulated that it might improve outcome in severe malaria. The next phase of trials investigated the addition of Pt to standard antimalarial therapies, primarily aimed at suppression of TNFα production. In total, five trials investigated its use in cerebral malaria, or all-cause severe malaria -121. TwoA controlled factorial trial of monoclonal antibodies (Mab) against TNF-α in Gambian children (N = 610) which also compared artemether and quinine, showed faster fever clearance but no survival benefit in children treated with Mab (19.9%) compared to controls (20.8%), after adjustment for antimalarial strategy . The triin vitro and reduce sequestration of late-stage parasites in uncomplicated P. falciparum malaria [A controlled Phase II trial investigating the use of the oral anthelmintic drug levamisole hydrochloride  include malaria . Linked P = 0.006) [P = 0.733) [P = 0.489). In survivors rates of neurologic sequelae at three months were the same (10%) in both arms [Since convulsions are common in severe malaria, in particular the sub-group with cerebral malaria, and associated with adverse outcomes including death and neurological sequelae (in children), routine administration of anti-epileptics to prevent the seizures was suggested. In a double-blind controlled trial in Thailand involving both children and adults with cerebral malaria, randomised to receive either a single dose of phenobarbitone (Pb) or placebo, there was a lower seizure rate in the Pb group at 12.5% (3/24) compared to 54% (13/24) in the placebo arm (= 0.006) . There w= 0.006) . The chi= 0.733) . In the oth arms . A studyoth arms .Leading up to these trials were two observational studies, one case series and one uncontrolled trial, investigating mannitol and other osmotic diuretics as adjuncts for treating brain swelling  in African children with cerebral malaria ,131. TheThe use of iron chelating agents was considered for adjunctive therapy in severe malaria. The positive hypothesis suggested that by chelating free iron these drugs would thus withhold iron which is required by malaria parasites to mature and multiply and, thus, limit or inhibit parasite reproduction rate. A Cochrane review of the data on the uses of iron chelation was conducted in 2007 . Whilst The first study conducted in a Zambian hospital involving 83 children with cerebral malaria indicated a lower mortality in DFO recipients, 7/42 (16.7%), compared to placebo, 9/41 (22%). Moreover, time to coma recovery was faster in the DFO arm . A seconP = 0.011), as was median coma resolution although with wide variation and, thus, was non-significant. Need for renal replacement therapy (two in each arm) or death (arm in each arm) were similar [P = 0.74) or coma recovery times (P = 0.46). Parasite clearance time was increased but largely attributable to differences in baseline parasitaemia,nor did NAC have any effect on red cell deformability properties [N-acetylcysteine (NAC) has been proposed to lead to correction of metabolic acidosis by its antioxidant effect acting through direct scavenging of free radicals and replenishment of glutathione and cysteine. NAC has also been shown to have anti-parasitic effects . Three t similar . A place similar . A multioperties . In summoperties .Both dopamine and adrenaline (epinephrine) are used in the management of infection-induced haemodynamic shock as second line treatments when shock is refractory to fluid boluses or when hypotension prevails . DopaminP = 0.004). There was a non-significant increase in neurological sequelae in survivors in those treated with albumin 9/97 (9%) compared to other resuscitation fluids, 6/54 (6%) in saline and 1/37 (3%) in gelofusine [Justification for investigating fluid resuscitation as an adjunctive treatment in children with severe malaria originated from the high mortality noted in association with metabolic acidosis ,67,144. lofusine . A furthlofusine .P. falciparum malaria . The data monitoring committee stopped the trial early after enrollment of 3,141 of a projected 3,600 due to safety concerns. The primary endpoint, 48-hour mortality, was significantly worse in the two fluid bolus arms, 111/1,050 (10.6%) in the albumin-bolus and 110/1,047 (10.5%) in the saline-bolus, compared to the control (no bolus) 76/1,044 (7.3%); relative risk (95% CI) any bolus versus control 1.45 . Secondary endpoints including 28-day mortality were worse in the bolus arms, 12% versus 8.7% in the control arm. There was no difference between the treatment arms in the number of adverse events . In the subgroup with severe malaria, 48-hour mortality was 110/1,202 (9.2%) in the bolus arms compared to 34/591 (5.7%) in the control (no bolus) arm, a relative risk of death of 1.59 (1.10 to 2.31). In all pre-specified sub-groups analysis, there was no evidence of a benefit of fluid boluses [P <0.001). Despite this, excess mortality with boluses was still evident in both ‘responders’ (relative risk 1.98 (0.94 to 4.17), P = 0.06), as well as ‘non-responders’ at one hour (relative risk 1.67 (1.23 to 2.28), P = 0.001), with no evidence of heterogeneity (P = 0.68) [P = 0.008), rather than respiratory events or neurological terminal clinical events [P = 0.07 by two-sided Fisher’s exact test). Moderate hypotension, consistent with many definitions in paediatric shock, was also associated with increased 48-hour mortality on boluses (an absolute risk of 9.4% (‐2.6 to 21.4%)) [The Fluid Resuscitation as A Supportive Therapy trial (FEAST) was conducted to provide evidence on the best fluid management for children with shock and severe febrile illness for doctors practicing in hospitals in resource-limited settings . FEAST w boluses . In a su = 0.68) . Examinil events . A major 21.4%)) .A systematic review conducted after the FEAST trial concluded that the majority of evidence from randomised trials to date came from the FEAST trial and that boluses significantly increased mortality of children in shock, compared to those who did not receive boluses. The evidence was considered to be of high quality and sufficiently precise . Prior tA Cochrane review including the only two African randomised controlled trials (RCTs) ,159 condIn conclusion, human trials carried out on the basis of pathophysiology studies, have so far made little progress on reducing mortality, despite appearing to reduce morbidity endpoints ; more often than not, the supportive interventions have shown harm. A new line of investigation that aims to disrupt sequestration  looks pr


The diagnosis of malaria during pregnancy is complicated by placental sequestration, asymptomatic infection, and low-density peripheral parasitaemia. Where intermittent preventive treatment (IPT) with sulfadoxine-pyrimethamine is threatened by drug resistance, or is inappropriate due to low transmission, intermittent screening and treatment (ISTp) with rapid diagnostic tests for malaria (RDT) could be a valuable alternative. Therefore, the accuracy of RDTs to detect peripheral and placental infection was assessed in a declining transmission setting in Papua New Guinea (PNG).Plasmodium lactate dehydrogenase (pLDH), and light microscopy (LM), to diagnose peripheral Plasmodium falciparum and Plasmodium vivax infections during pregnancy, were assessed using quantitative real-time PCR (qPCR) as the reference standard. Participants in a malaria prevention trial in PNG with a haemoglobin ≤90 g/L, or symptoms suggestive of malaria, were tested. Ability of RDT and LM to detect active placental infection on histology was evaluated in some participants.The performance of a combination RDT detecting histidine-rich protein-2 (HRP-2) and P. falciparum, 19 P. vivax, 6 mixed infections). Overall, RDT detected peripheral P. falciparum infection with 45.6 % sensitivity (95 % CI 38.0–53.4), a specificity of 96.4 % (95.0–97.4), a positive predictive value of 68.4 % (59.1–76.8), and a negative predictive value of 91.1 % (89.2–92.8). RDT performance to detect P. falciparum was inferior to LM, more so amongst anaemic women  compared to symptomatic women . RDT and LM missed 88.0 % (22/25) and 76.0 % (19/25) of P. vivax infections, respectively. In a subset of women tested at delivery and who had placental histology (n = 158) active placental infection was present in 19.6 %: all three peripheral blood infection detection methods  missed >50 % of these infections.Among 876 women, 1162 RDTs were undertaken . qPCR detected peripheral infection during 190 RDT episodes  women. These findings have implications for the management of malaria in pregnancy. The adverse impact of infections undetected by RDT or LM on pregnancy outcomes needs further evaluation.In PNG, HRP-2/pLDH RDTs may be useful to diagnose peripheral The online version of this article (doi:10.1186/s12936-015-0927-5) contains supplementary material, which is available to authorized users. Malaria during pregnancy is a preventable and treatable disease, which remains responsible for an enormous health, social and economic burden for communities in the developing world. Malaria infection is a major threat to maternal and neonatal survival, resulting in up to 200,000 infant deaths and 10,000 maternal deaths each year [Plasmodium falciparum-infected erythrocytes in the placental intervillous space, termed placental malaria (PM) [Diagnosing malarial infection in pregnancy is challenging. In areas of high endemicity the majority of infected women are asymptomatic . Diagnosria (PM) . Antenatria (PM) , yet it ria (PM) . Intermiria (PM) , overcomria (PM) , 8, and ria (PM) , and as ria (PM) .P. falciparum infections in causing adverse pregnancy outcomes, in particular anaemia, in some [Recently, there has been considerable interest in intermittent screening and treatment in pregnancy (ISTp) as an alternative to SP-IPTp, in particular in areas where high-level drug resistance is a concern . This ap in some , 12–14,  in some , 16, stuP. falciparum and P. vivax frequently co-exist [Plasmodium knowlesi, are sympatric in PNG, combination RDTs detecting both P. falciparum-histidine-rich-protein-2 (HRP-2) and the genus-specific malaria antigen lactate dehydrogenase (pLDH) are most appropriate, and were shown to be appropriate for malaria treatment amongst febrile children in PNG [RDTs perform well outside of pregnancy and the WHO recommends their use for the diagnosis of malaria in this context . Less isco-exist –20 and cco-exist . In Papuco-exist , 23. PNGco-exist . Given an in PNG . To dateP. falciparum and P. vivax infections as detected by qPCR in the context of a large clinical trial of malaria prevention in pregnancy in PNG. Secondary objectives included an assessment of RDT performance characteristics relative to qPCR amongst both women with asymptomatic anaemia vs those with suspected malaria, and an evaluation of the performance of peripheral blood RDT, LM and qPCR to detect placental infection as observed on histological examination.This study evaluated the accuracy of an HRP2/pLDH combination RDT and LM to diagnose peripheral P. falciparum and P. vivax [This study was conducted between July 2010 and November 2013 in a prospective cohort of pregnant women enrolled in a clinical trial evaluating three doses of IPTp with SP plus azithromycin vs a single dose SP plus chloroquine and two placebo doses from second trimester in PNG (NCT01136850) . The triP. vivax . PrevaleP. vivax .P. falciparum [During the original trial passive case detection forms (morbidity episodes) were completed for women who reported new or recent illness or where found to be anaemic. The trial was designed such that an RDT was performed during a morbidity episode when malaria was suspected clinically or when women were found to be moderately or severely anaemic  <90 g/L for moderate, and <60 g/L for severe  and/or presence of pallor. Because women living in lowland PNG were highly likely to be at least mildly anaemic by WHO standards , a more lciparum , 31.P. falciparum and P. vivax parasitaemia was assessed using qPCR as the Ref. [Accuracy of the RDT, and LM, to detect peripheral the Ref. . A subsePregnant women presenting for their first antenatal visit were screened according to inclusion criteria of the original trial . In brieP. falciparum-specific malaria antigen HRP-2, the other genus-specific LDH. Using LM as reference, the manufacturer reported sensitivities of 98 and 96 % to detect peripheral P. falciparum and P. vivax infections, respectively in non-pregnant individuals [Malaria was diagnosed using the commercially available three-band CareStart™ P.f/Pan combo RDT , which is pre-coated with two monoclonal antibodies, one detecting ividuals .Testing was undertaken by trained clinical research staff, and test kits were stored and performed according to the manufacturer’s instructions . There wPeripheral blood smears were prepared, and venous blood samples taken, each time an RDT was performed. Labeled blood smears were air-dried and stained with 4 % Giemsa for 30 min. Thick smears were used to count the number of asexual parasites per 200 leukocytes (or per 500 if <10 parasites/200 leukocytes), assuming 8000 leukocytes/µL of blood; slides were declared negative if no parasite was seen in 200 oil-immersion fields. Two microscopists read each slide, and third reads were performed to resolve discrepant results . If specP. falciparum and P. vivax infections in peripheral and placental blood samples using an established methodology with a sensitivity of 1 parasite per microliter [Plasmodium ovale and Plasmodium malariae infections in pregnant PNG women are rare [qPCR was performed to detect croliter . Plasmodare rare , and budA subset of women had placental tissue collected for histological processing and analysis as described previously . PlacentReaders of all aforementioned the index tests (LM and RDT) and respective reference standards  were blinded to the results of the other tests.P. falciparum: P. vivax has been associated with placental changes but these differ markedly from P. falciparum and are rare [qPCR was used as the reference method for the detection of peripheral parasitaemia, and placental histology to identify women with active placental infection. All active placental infections were assumed to be due to are rare .P. falciparum if showing (a) a positive reaction at the HRP-2 band, or (b) positive reactions at both the HRP-2 band and pLDH band. The RDT was classified positive for P. vivax if showing a positive reaction at the pLDH band only, or at both the HRP-2 band and pLDH band (possible mixed infection).An RDT was classified positive for t test and the Mann–Whitney-U test for parametric data and nonparametric data, respectively.Data were double entered into a study databases  and analysed using Stata 12.0 . Univariate comparisons of variables were undertaken using the Chi-squared and Fisher’s exact test for binary data, as appropriate, and the Student’s P. falciparum parasitaemia, and RDT, LM and qPCR of peripheral blood for the detection of active placental infection. Analysis of diagnostic performance of RDT and LM to detect P. vivax infection was restricted to sensitivities, as qPCR did not include P. ovale and P. malariae. In addition, performance of RDT to detect malarial infection (any species) observed on LM (the current reference standard in PNG) was assessed. Sensitivities and specificities were compared using Liddell’s exact test for pairwise comparisons of proportions [Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) with 95 % confidence intervals were calculated for the comparisons of RDT against qPCR, and LM against qPCR, for the detection of peripheral portions , 42. Parportions . The stuWritten informed consent was obtained from all participating women. Study procedures were in accordance with good clinical and laboratory practice. Ethical approval for study protocol was obtained from the PNG Institute of Medical Research Institutional Review Board (0815), the PNG Medical Research Advisory Council (08.01), and the Melbourne Health Human Research Ethics Committee (2008.162). The trial was registered with the United States National Institutes of Health Clinical Trials Registry (NCT01136850) and has been reported according to STARD guidelines.Of 2793 women enrolled in the original trial, 876 31.4 %) women met the criteria for RDT testing at least once during pregnancy, had complete data for peripheral blood RDT, LM and qPCR, and were thus eligible for inclusion in the present analysis. The remaining 1917 were either withdrawn from the original study , did not meet testing criteria , or had incomplete test results  a total of 1162 tests were performed. One quarter of women  had two or more RDT screening episodes: the mean  number of days between testing episodes was 57  days. A quarter of tests were performed because women presented with symptoms suggestive of malaria   . Compared with women tested due to anaemia, women who presented with symptoms of suspected malaria were more likely to have a positive RDT and to have P. falciparum infection diagnosed by LM or qPCR, and had higher parasite densities when they were infected  RDT screening episodes , and a higher proportion  of infections were missed in women who were asymptomatic   ; although LM missed more than three quarter of infections it detected twice as many compared to RDT . The geometric mean density of these infections was 157 parasites/μL , with seven out of nine infections exhibiting a parasite density <200 parasites/μL. Specificities were not assessed as the RDT measured pan-specific LDH and qPCR for P. ovale and P. malariae were not performed.Prevalence of peripheral  low n = 5; althouA total of 49 of 143 (34.3 %) infections of any species detected by LM remained undetected by the HRP-2/pLDH RDT. Geometric mean density of these infections was 278 parasites/μL . Specifically, RDT had a sensitivity of 66.0 % (95 % CI 57.6–73.7), specificity of 96.1 % (94.7–97.2), PPV of 70.4 %  and NPV of 95.2 % (93.7–96.4) to detect microscopic peripheral parasitaemia.P. falciparum by RDT. RDT on peripheral blood failed to detect more than half of the women with active placental infection, as did LM and qPCR (Table P = 0.060).A quarter of women (221/876) had an RDT performed at delivery. Of these 71.5 % (n = 158) had a placental biopsy taken for histological evaluation of placental malaria, and 31 (19.6 %) had evidence of active infection  detected by LM, falling well below the WHO performance criterion of 90 % sensitivity, considered to indicate adequate performance [P. falciparum was higher amongst symptomatic women, and poorer for asymptomatic anaemic women, or in women infected with P. vivax. In line with previous findings [P. falciparum and P. vivax infections during pregnancy.The performance characteristics and utility of an HRP-2/pLDH combination RDT to detect parasitaemia in pregnant women in coastal PNG were evaluated as part of a clinical trial providing insecticide-treated bed nets and at least one dose of IPTp. RDTs were undertaken amongst trial participants presenting with anaemia or symptoms suggestive of malaria only. In this context RDT failed to diagnose about half of peripheral and placental formance . RDT perfindings , RDTs diP. falciparum as detected by qPCR suggests limited utility as a malaria screening tool in pregnancy in PNG. Poor performance observed in this study may be explained by low sensitivity to detect low density parasitaemias, although the median parasite densities for P. falciparum exceeded the minimum detection threshold documented in laboratory testing of the RDT test (>100/µL) [The overall poor performance of an HRP-2/pLDH combination RDT to detect >100/µL)  and thos>100/µL) . Not all>100/µL) . In Ugan>100/µL) . The difP. falciparum parasitaemia amongst asymptomatic women with anaemia (Hb <90 g/L). This finding has implications at a number of levels.It is well established that in areas with moderate-to-high transmission intensity the majority of parasitaemic pregnant women will be asymptomatic. As such, RDT performance to detect infection in asymptomatic women is critical. RDTs had poor sensitivity for detection of peripheral The suboptimal performance of the HRP-2/pLDH RDT amongst asymptomatic anaemic women suggests that it may not be appropriate to use this diagnostic tool as a component of IST. Although the association between infections missed by RDT and adverse mother-infant outcomes could not be evaluated in the present study, a recent study conducted in Benin provides compelling evidence of the detrimental effect of submicroscopic infections on increased risk of maternal anaemia and low birth weight in primigravidae . Second,Taken together these findings suggest that further optimization of existing RDTs, or the development of novel, molecular approaches such as loop-mediated isothermal amplification (LAMP) , 48, to RDT performance was better in women screened because of malaria symptoms than for asymptomatic anaemia, possibly because of the higher parasite densities in symptomatic women. This observation is in keeping with previous research that demonstrates near-adequate performance in febrile pregnant women in Africa . The preAt delivery, all measures of parasitaemia in peripheral blood significantly underestimated active placental malaria by histology, probably due to occult placental sequestration. This supports a previous study by Mayor et al. , who shoP. vivax infection in non-pregnant individuals report varying performance [P. vivax infections was lower than expected, and it was a rare cause of clinical malaria in the present study. The limited data show that RDT or LM missed nearly three quarters of infections detected by PCR. This suggests it is not a good screening tool for P. vivax infection in pregnancy. Future studies including PCR data to differentiate potential P. ovale and P. malariae infections, which are present but rare [P. vivax infection.The few studies to evaluate RDT for formance , 20, 44.but rare , 51, areAlthough this study is one the largest to assess the usefulness of an HRP-2/pLDH RDT in pregnancy in the Asia–Pacific region, it is subject to a number of limitations. First, the findings are restricted to pregnant women presenting with moderate to severe anaemia and/or non-specific clinical malaria symptoms: RDT performance amongst asymptomatic pregnant women without anaemia is unknown. Second, only a subset of women were tested at delivery and had placental biopsies taken: the small sample size increases the risk of random error and does not allow definitive conclusions regarding the performance of HRP-2/pLDH-RDT to detect placental infection. In addition, histology was not evaluated against placental blood qPCR. Third, the low prevalence of infection overall limits extrapolation of findings to areas of higher malaria transmission, but does reflect much of the malaria epidemiology across the Asia–Pacific region. Fourth, this study was conducted within the context of an IPTp trial in women sleeping under insecticide treated bet nets. Although most RDT screening events took place at enrolment, before women received any treatment, interpretation of the study data is limited to this context. Moreover, qPCR species-correction of five infection episodes may have biased the diagnostic performance of LM. While operator error cannot be excluded at clinical or laboratory level, clinical training, refresher training and competency assessment were provided throughout the study, rendering operator factors as an unlikely cause of poor RDT performance. Microscopy and molecular diagnostic facilities at the PNG Institute of Medical Research undergo regular external quality control and operate at the highest standard. Finally, the low prevalence of malaria in this cohort and the study design did not permit an evaluation of the impact of missed infections on pregnancy outcomes.P. falciparum infections in symptomatic pregnant women, but should not be employed for screening amongst asymptomatic (anaemic) women. A more sensitive tool is required to effectively detect and guide treatment of all women with Plasmodium spp infections. The potential adverse impact of infections undetected by RDT or LM on pregnancy outcomes needs further evaluation. Until more sensitive point of care diagnostics are available, IPTp with SP is likely to remain the optimal strategy for malaria prevention in pregnancy in PNG.At the current level of transmission in lowland coastal PNG, HRP-2/pLDH RDTs may be used to diagnose peripheral