Plasmodium falciparum histidine-rich protein 2 gene deletion has been reported in India. Such isolates are prone to selective transmission and thus form a challenge to case management. As most of the rapid malaria diagnostic tests are based on the detection of HRP2 protein in the blood, we attempted to use Glutamate Dehydrogenase (GDH) as a biomarker for the diagnosis of P. falciparum. Recombinant PfGDH was successfully cloned, expressed and purified using the Ni-NTA approach. Polyclonal antibodies were raised against full-length rPfGDH and its peptides. Antibodies for rPfGDH showed a strong immune response against the recombinant protein. However, antibody showed no affinity towards the peptides, which suggests they failed as antigen. Antibodies for rPfGDH significantly detected the GDH in human blood specimens. This is the first report where P. falciparum GDH was detected in malaria cases from various parts of India. The raised polyclonal antibodies had shown an affinity for PfGDH in quantitative ELISA and are capable to be exploited for RDTs. This research needs further statistical validation on a large number and different sample types from candidates infected with P. falciparum and other species.In recent years,  For malaria diagnosis, different invasive methods are broadly in use. Slide microscopy remains the gold standard to identify the parasite and their load in malaria patient. Molecular biology based approaches are highly sensitive and accurate but are not suitable for rural areas due to high cost, complex methodologies, expensive equipments and skilled manpower. However, the invasive methods always have concerns such as fear of contacting with other blood related diseases, pain associated with finger pricking, proper disposal of needles, correct interpretation of results and adherence to hygiene practices3. In non-invasive approach saliva collection is feasible, to check the presence of parasitic biomarkers using rapid diagnosis test (RDT) and may serve as an excellent surveillance tool for malaria elimination programme2. Whole saliva samples from children with uncomplicated malaria showed 77.9% sensitivity against 97.9% from blood and 48.4% from supernatant of spun saliva samples, using lactate dehydrogenase (LDH) RDT and genotyping of P. falciparum4.In the era of malaria elimination, several African and South East Asian countries are pushing their limits to be free from malaria. However, these countries are facing the challenges related with malaria diagnosis. In South East Asia region, India accounts for about 80% of malaria cases and 60% deaths due to malariahrp2 and hrp3 single or dual gene deletions were reported from Indian P. falciparum isolates and other parts of the world7. Bharti et al. has evaluated more than 20 brands of RDT based on HRP2 and their sensitivity varied between 80–97% for P. falciparum8. Deletion of hrp2/3 gene in P. falciparum raises a concern for the national programme as HRP2 based RDT’s are broadly used for malaria diagnosis, this may result in misdiagnosis and false treatment strategies. A recent study showed that in eight malaria endemic Indian states P. falciparum Glutamate dehydrogenase (GDH) is genetically conserved and is under negative selection as observed by tajima D test9. GDH can be exploited as a potential marker for detection of Plasmodium falciparum.Antigen based RDT kits, available commercially lacks in the expected specificity and sensitivity Table . In past10. GDH is approximately 50–60 kDa sized metabolic soluble protein12. In P. falciparum three gdh genes encoding potential GDH proteins are present, two genes are on chromosome 14  and one gene on chromosome 8 14. Glutamate dehydrogenase was first isolated and characterized from the plasma of Plasmodium falciparum infected malaria patients12. GDH is a heat resistant and soluble antigen which can be used for antibodies production to improve immunodiagnostic assays16. Malaria parasite shows similar metabolism as host but the characteristics of the GDH enzyme are different based on their kinetical, electrophoretically, specificity of co-factors, substrates, degree of affinity, and immunogenicity. Reason behind the selection of Glutamate dehydrogenase is the exhibition of NADP-specific GDH activity. Malaria parasite secretes NADPH in combination with NADP-specific isocitrate dehydrogenase, which is not found in host red blood cells. Reports had shown that the GDH from a) animal origin requires purine nucleotide and b) P. chabaudi  does not require purine nucleotides17. GDH is one of the P. falciparum enzymatic antigen which has been immuno-detected by the antibodies raised in animals or from the sera of P. falciparum malaria patients from Yanumana Amerindians living in Venezuela12. Also, a recent study showed that Glutamate dehydrogenase gene sequence is highly conserved in P. vivax and used for diagnosis of vivax malaria in South Korea. Antibodies against PvGDH did not cross react with the sera from P. falciparum positive patients. Seol et al. suggested that GDH could be used as a potential antigen for seroepidemiology studies18. Therefore, we attempted to investigate P. falciparum GDH as a marker protein for malaria parasites20. In this article, PfGDH in blood sample from Indian malaria patients was investigated.Malaria parasites uses GDH as crucial enzyme to obtain energy via Krebs cycle, where it oxidizes glutamate to alpha-ketoglutarate utilizing NADP and releasing NADPH during intraerythrocytic stagepfgdh) of P. falciparum 3D7 located on chromosome 14 was successfully expressed in BL21(DE3) E. coli cells. rPfGDH was purified more than 90% with Ni-IMAC and analyzed on SDS-PAGE and confirmed by Western Blot.The glutamate dehydrogenase gene  were used to check the response of the polyclonal antibodies. It was found that only antibodies against full length rPfGDH has shown affinity, wells with less concentration of protein has shown maximum binding. Results showed that none of the predicted PfGDH peptides are eligible for P. falciparum and P. vivax validated by microscopy and PCR) were screened to detect the presence of Pf glutamate dehydrogenase by direct ELISA method. Out of twenty-four positive samples, 23 samples showed binding with the rPfGDH antibodies. On the other side, the negative samples did not show any significant response against the antibodies. All the experiments were performed in triplicate and were repeated to confirm the assay outcome. The cut-off value was the mean +2 SD of negative control (sample from uninfected human blood). The geometric mean of responses recorded from above samples showed significant outcome as the value of P < 0.000121  (upto 200 parasites/μl), dried blood spot (n = 5), plasma (n = 4) and serum samples (n = 4) and known negative specimens (n = 14) for P. falciparum (n = 8) and positive P. vivax (n = 6). The blood specimens with no P. falciparum (diagnosed with microscopy and PCR) were used as negative controls in Quality assurance laboratory at ICMR-NIMR, a WHO recognized facility.We have analysed total 27 anonymized known malaria samples, that included known positive We analysed anti-PfGDH antibodies for sensitivity and specificity using MedCalc statistical online software. The anti-PfGDH antibodies are 96.30% sensitive and 100% specific to PfGDH antigen, where the samples were true positive (n = 26), false negative (n = 1); true negative (n = 14) and false positive (n = 0).All study sample subgroups were compared and shown in table no. 2 . InfecteThe presence of GDH antigen in blood specimen collected from malaria patients was detected Fig. . A varioThe anti-GDH antibodies showed strong sensitivity (96.30%) and specificity 100%) towards PfGDH antigen  such as cross-reactivity against different epitopes, variability due to different animals and time, and to improve the sensitivity of the assay, attempts are in progress to develop monoclonal antibodies (MAb) against the recombinant PfGDH. The MAb will enhance the reproducibility, sensitivity and reduce the background noise. MAb are preferred over PAb to develop the assay into the RDT kits.hrp2 gene deletion has been observed7. HRP2/3 based RDT kits are commercially available and a key tool in malaria diagnosis before the treatment is given. The significant challenges associated with the currently available RDT kits are their maintenance and stability in fields which are remotely located and have very less or no resources to maintain the quality of these kits. These issues substantially affect the quality of the RDT results, which leads to misdiagnosis and implementation of wrong treatment or management of the disease.In recent years, increase in the prevalence of hrp2 gene deletion is a challenge and there is an urgent need to identify, develop and bring a new biomarker with the robustness to resist against the gene deletion in parasitic genome and overcome other issues. Since past few years, several researchers has been indicating towards Glutamate dehydrogenase protein which have potential to be developed as a biomarker to detect malaria parasite23. PfGDH protein encoded gene can stay tough in the field as it is present in three copy number on chromosome 8 and 1414. In a recent study from India, the authors found PfGDH as genetically conserved in eight highly malaria endemic states9. PfGDH is also heat resistant16 and could be exploited in RDT kits for real time field conditions, where logistics, temperature and humidity are the major challenges.To overcome et al.24 reported that polyclonal antibodies raised against P. knowlesi were reacted with NADP specific glutamate dehydrogenase of P. knowlesi, P. falciparum and Proteus spp. Similarly, monoclonal antibodies raised against GDH from Proteus spp. were cross-reacted with P. falciparum. Further GDH is known to be specific to malaria parasites22, this observation is concurrent with PfGDH antibodies which showed discrimination with P. vivax samples. This could be due to the distinct epitope region, other than substrate - enzyme binding site22. In this pilot study PfGDH was detected in samples with low parasitemia (upto 200 parasites/μl). Our findings are supported by the outcomes from a recent study where PvGDH was detected in samples with low (<1000) and high (>10000) parasitemia count without any significant difference18. This data potentiates the hypothesis of detecting the GDH antigen in soluble form in low parasitemic samples.Ling 18. With respect to the infected RBC and eluted DBS samples no significant difference was observed, which suggests that both sample types are equally capable to detect the PfGDH antigen.Blood sample subgroups comparison showed significant outcomes. Anti-rPfGDH antibodies showed strong response with the plasma, which confirms the presence of soluble GDHPlasmodium species infected blood samples, we performed assay with P. vivax samples using anti-rPfGDH antibodies and interestingly there was no cross-reactivity observed. It was also noticed that anti-PfGDH antibodies show no cross-reaction with any of the human enzymes, similar observation was made by Rodriguez-Acosta et al. when they characterized the hyper immune sera against PfGDH from Yanumana Amerindians12 With these promising results, in future blood specimens infected with P. vivax, P. malariae, P. ovale and P. knowlesi from different endemic regions of India will be tested. We will attempt to perform the study on samples with low to high parasitemia to check to lower and higher limits of anti-PfGDH antibodies sensitivity. The outcome from those studies will help us to further optimize and strengthen the development of PfGDH antigen as biomarker for malaria diagnosis.Due to non-availability of other P. falciparum infection confirmed by microscopy and RDT in form of dried blood spot, whole blood, plasma and serum, preserved at ICMR-National Institute of Malaria Research, New Delhi, India.The study was approved by the Institutional Human Ethics Committee (reference no. ECR/NIMR/EC/2017/64) to use already collected anonymized specimens with To perform animal experiments, the study was approved by Institutional and Animal Ethics Committee (reference no. IAEC/NIMR/2018-2/10) following the national guidelines from The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA Registration No. 33/GO/ReBi/S/99/CPCSEA), Government of India.pfgdh) of 1413 bp  was PCR amplified using the forward primer 5′- CGGCATATGAGTGCTCTTAAAGACAAAACGG-3′ (the NdeI restriction enzyme site is underlined) and reverse primer 5′-CGGCTCGAGTCAATGATGGTGATGGTGATGACAACCTTGTTC-3′ . Molecular recipe to amplify GDH gene included 0.25U of Q5 High Fidelity DNA polymerase, 1x Q5 reaction mixture and 1x Q5 GC content enhancer , 2 ng gDNA isolated from Plasmodium falciparum positive blood sample, 1 μM of each primer and 2 μM dNTP’s  in total 25 μl reaction volume. The thermal profile to amplify GDH gene was 98 °C-30 sec, followed by 30x cycle of 98 °C-7 sec, 55 °C-20sec, 72 °C-1 min and final elongation for 2 min at 72 °C, with 10 °C as storing condition. PCR amplified products were observed in 1% agarose gel run in 0.5x TBE buffer under UV transilluminator  and cloned into the pET22b+ expression vector  E. coli cells as per manufacturers recommended protocol. The clone designated with rPfGDH the expresses the protein of interest  was selected for further studies.The amplified PCR product was digested using tor Fig.   and the culture was further incubated for 6 h at 37 °C at 225 rpm. Subsequently, the culture was centrifuged at 8000 g for 10 min to harvest the cells and stored at −20 °C. Later, cell pellet was resuspended in 20 ml GenePro Total Protein isolation buffer  and incubated on ice for 10 min and centrifuged at 16000 g for 10 min. The clear cell lysate was collected and mixed with 1.5 M GuHCl and passed through 2 ml bed volume of cOmplete His-Tag purification resin  using gravity flow columns . The column was washed with 4 bed volumes of buffer containing 40 Tris-HCl pH 7.4 and 20 mM and 50 mM Imidazole. rPfGDH was eluted with 100 and 150 mM imidazole, both eluates were mixed and passed through ultra-centrifugal device with a cut-off of 30 Kda  at 5000 g for 10 min at 4 °C. The purification procedure was monitored by SDS-PAGE denaturing conditions, where the rPfGDH was appeared approximately as a ~52 kDa, which was in the agreement with its theoretical molecular mass  supplemented with 100 μg/ml ampicillin to an ODass Fig. . The rPfass Fig. . The rPfhttp://tools.iedb.org/bcell/) using hidden Markov model and propensity scale method was used to predict epitopes. The four dominant peptides considered for this study with high immunogenicity score, linear epitope region and hydrophilicity were: a) MGGGKGGSDFDPKGKSDN, b) PCTDVPAGDIGVGGR, c) NEQYSSDKYFPTFEET, d) PFQQGKLRKNGGVPHD. All the peptides were synthesized by GL Biochem Ltd, Shanghai, China.Bepipred Linear Epitope Prediction an online tool , purchased from ICMR-National Animal Resource Facility for Biomedical Research, Hyderabad and kept at in-house animal facility of NIMR for the duration of experiments.On day one each mouse (five mice per group) was immunized with 100 μg antigen (30 μl) (rPfGDH and peptides) mixed with equal volume of Complete Freund’s Adjuvant  at subcutaneous site using 30 g x5/16 U-40 syringe (Dispovan). Subsequently, two booster doses were given on day 21 and day 42 with 50 μg (30 μl) antigen in Incomplete Freund’s Adjuvant . Control mice were injected with 1xPBS and the respective adjuvant. On day 50, first bleed approximately 100 μl was collected from tail vein per mouse. All the animals were given with third and fourth booster doses on day 57 and day 71, second bleed was collected on day 79. From blood serum was separated after incubating the it at 37 °C for 30 min followed by a centrifugation at 2500 rpm for 5 min and stored at −20 °C for later use.Polyclonal antibodies raised against full length PfGDH and above mentioned peptides were purified using Nab Spin Columns  by following the recommended protocol from the manufacturer. 100 μl of serum sample was mixed with the resin in the columns and was washed three times with binding buffer and subsequently eluted with 400 μl of elution buffer. Purified samples were aliquoted and stored at −20 °C for downstream experiments.2SO4. The plate was read at 450 nm on Spectrostar ELISA plate reader. Animal sera without rPFGDH was used as negative control.rPfGDH and peptides (100 μl of a 1 μg/ml solution in 1xPBS) was coated in the wells of high binding 96 well MaxiSorp flat bottom plate  and incubated over night at 4 °C. The plate was washed with 1xPBS three times and blocked with 2.5% BSA in 1xPBS for 1 hr at 37 °C. Following three washes with 1xPBST (0.2% Tween 20) and 1xPBS, the plate was incubated for 1 hr at 37 °C with 1:500 dilution of anti-rPfGDH and anti-peptides serum. The plate was then washed three time with 1xPBST and 1xPBS each. The bound anti-rPfGDH and anti-peptides were detected by goat-anti-mouse IgG-HRP antibodies (plate was washed after each step with 1xPBST and 1xPBS). Subsequently, the antigen-antibody reaction was developed with ELISA TMB substrate (KPL) for 20 min in dark at 37 °C and the reaction was stopped with 0.2 M HThree 3 mm discs from filter paper dried blood spots were punched out in 200μl of 1x phosphate buffered saline (PBS) containing 0.05% tween 20 and were incubated overnight at 37 °C. The following day, samples were centrifuged at 8000 g for 10 minutes and supernatant containing serum content was stored at −20 °C until further use.2SO4 was used to stop the reaction. The results were recorded at 450 nm on Spectrostar ELISA plate reader. Prism 8.0 was used to analyse and plot the data21  was coated in triplicate and incubated overnight at 4 °C. The following day, wells were washed with 1xPBST  and blocked with 2.5% BSA (Sigma) in 1xPBS (GCC Biotech) and incubated for 1 hr at 37 °C. The wells were washed thrice with 1xPBST and added with purified PfGDH antibodies in 1:1000 dilution and incubated for 3 hrs at 37 °C. Subsequently, wells were washed thrice as mentioned above. To detect PfGDH antibodies, goat-anti-mouse IgG-HRP antibodies added in wells in 1:5000 dilution and incubated for 45 min at 37 °C and wells were washed as described previously. In each well 100μl of TMB substrate (KPL) was added and incubated for 15 min in dark at 37 °C and 0.2 M HP. falciparum in blood sample. However, present work will be further validated on large number and on different sample types including whole blood, serum, eluted DBS, saliva and urine from malaria patients infected with P. falciparum and other species.Preliminary results from quantitative ELISA using antibodies against PfGDH had shown strong affinity, sensitivity and specificity towards the GDH antigen from malaria patient. This is a novel work and was not reported by anyone on malaria cases from India. The results are promising to utilise GDH as a potential biomarker to detect Supplementary Information.


Adverse outcomes  were more common in fetuses and neonates from mothers with prolonged viremia (40.0%) compared with those from infected mothers without prolonged viremia  or those from noninfected mothers . Congenital infections were confirmed more often in fetuses and neonates from mothers with prolonged viremia compared with the other 2 groups .Whether prolonged maternal viremia after Zika virus infection represents a risk factor for maternal–fetal transmission and subsequent adverse outcomes remains unclear. In this prospective cohort study in French Guiana, we enrolled Zika virus–infected pregnant women with a positive PCR result at inclusion and noninfected pregnant women; both groups underwent serologic testing in each trimester and at delivery during January–July 2016. Prolonged viremia was defined as ongoing virus detection  The recent worldwide epidemic confirmed maternal–fetal transmission of Zika virus (ZIKV) and its association with adverse perinatal outcomes, particularly severe central nervous system lesions and fetal losses  during the acute phase of infection. ZIKV viremia usually lasts from 2 days before to 16 days after symptom onset; median time of ZIKV RNA clearance is 5 days  compared with infected pregnant women without prolonged viremia and noninfected pregnant women. We also compared the rates of congenital infections between these groups.The study was conducted at the Centre Hospitalier de l’Ouest Guyanais  during January 1–July 15, 2016, at the beginning of the ZIKV epidemic. Persons for inclusion were initially identified either through routine serologic testing of all pregnant women admitted to the prenatal diagnosis unit of CHOG (performed in each trimester of pregnancy and at birth), or through serologic and molecular testing in cases of maternal symptoms, acute exposure in the previous 2 weeks , fetal or neonatal central nervous system anomalies, or in cases of amniocentesis performed for suspected CZS or other indications . All pregnant women with ZIKV testing available with ongoing follow-up at CHOG were invited to participate in the study. Written consent was obtained from all participants. The study received ethics approval from the CHOG institutional review board , as recommended by health authorities in France and other organizations  and testing for congenital ZIKV infection were performed for all neonates. Biologic, ophthalmologic, and imaging follow-up was offered for neonates from ZIKV-infected pregnant women  for RT-PCR, in-house IgM and IgG antibody-capture ELISA, and microneutralization assays for serologic testing. The limit of detection for serum samples tested using the Realstar Zika Kit was 0.61 (95% CI 0.39–1.27) copies/μL (t) value <37 was considered positive.Molecular and serologic testing was performed at the French Guiana National Reference Center for arboviruses  using the Realstar Zika Kit  or identification of ZIKV-specific IgM in the umbilical cord or neonatal blood or in cerebrospinal fluid. Details of congenital ZIKV testing are discussed elsewhere , or both. A confirmed congenital ZIKV infection was defined either by ZIKV RNA amplification by RT-PCR from Standardized differences were calculated to compare baseline characteristics of patients with prolonged viremia to those of the reference groups . These characteristics were considered unbalanced when the standardized difference was >0.15.Relative risks (RRs) and 95% CIs were calculated for fetal and neonatal adverse outcomes by using generalized linear regression. Adjusted RRs (aRRs) were calculated for variables reflecting unbalanced baseline characteristics that could represent confounding factors . When fetuses from mothers with prolonged viremia were compared with those from infected mothers without prolonged viremia, RRs were also adjusted for the trimester of maternal infection diagnosis. A robust SE option was used for twins in order to not affect the variance in considering twins as separate cases.We conducted a sensitivity analysis to test the robustness of our findings, using different criteria for the diagnosis of maternal prolonged viremia. We compared fetuses and neonates from patients with stable or increasing quantitative PCR (qPCR) values between the inclusion and the first follow-up with those from mothers with decreasing qPCR values.https://www.stata.com).The missing data were considered to be random, and thus we performed a complete case analysis. All statistical analyses were conducted by using Stata 15  with negative serologic test results for ZIKV were followed for routine scans at CHOG. Full maternal, placental, and neonatal testing was available for 326 of them (including 6 with twin pregnancies). These patients remained negative for ZIKV during the entire pregnancy and at delivery, and constituted the noninfected group. The recruitment process is summarized in As shown in Appendix 1 Table 3, baseline characteristics were similar between pregnant women with prolonged viremia and the reference groups, except for a history of congenital abnormalities or intrauterine fetal demise, maternal underlying conditions, rate of dichorionic twins, and high risk for fetal aneuploidy (>1/250), for which standardized differences >0.15 were observed. Maternal ZIKV infections were diagnosed earlier in pregnancies with prolonged viremia than in those without prolonged viremia.ZIKV testing and outcomes were available for 15 fetuses from 14 infected pregnant women with prolonged viremia (including 1 with a dichorionic twin pregnancy)  Table 4.Among 19 fetuses from mothers with an initially positive RT-PCR result without prolonged viremia, no fetal loss or termination of pregnancy was recorded. Neurologic anomalies were found in 1 (1/19 [5.3%]) of these fetuses, who also had ocular anomalies (confirmed at birth). Congenital ZIKV infections were confirmed in 5 (5/19 [26.3%]) of these newborns, of which 1 (1/5 [20%]) resulted in adverse outcomes (suspected CZS).ZIKV testing and outcomes were available for 332 fetuses or newborns from noninfected pregnant women (including 6 with dichorionic twin pregnancies). Four pregnancies (4/332 [1.2%]) resulted in termination of pregnancy . Four (4/332 [1.2%]) fetal losses were recorded. Among the fetuses with imaging studies and examination available, 17 (17/331 [5.1%]) had neurologic anomalies. None of these fetuses or neonates were found to be ZIKV-positive at birth.Overall, fetuses or neonates from mothers with a prolonged viremia during pregnancy exhibited a higher risk for adverse outcomes  compared with those from infected mothers without prolonged viremia  and noninfected mothers . Similar results were observed for fetal losses and neurologic anomalies when analyzed independently  Table 4.Congenital infections were confirmed more frequently in fetuses from mothers with prolonged viremia (9/15 [60.0%]) when compared with those from infected mothers without prolonged viremia  and noninfected mothers (0/332 [0.0%]). After adjustment for the trimester of maternal infection diagnosis and consideration of twin pregnancies in the variance, this trend persists (aRR 2.3 [95% CI 0.9–5.5])   only in the case of an abnormal prenatal ultrasound or symptoms at birth, in contrast to those from infected mothers who underwent routine postnatal imaging. However, when they are asymptomatic, some neonates from noninfected mothers might have undetected cerebral anomalies at birth, resulting in an overestimation of the consequences of prolonged Zika viremia. We cannot totally exclude this bias; however, all neonates from noninfected mothers underwent multiple prenatal ultrasound assessments (enhanced by 2 supplementary examinations with neurosonograms during the epidemic), reducing the risk for undetected cerebral anomalies.Molecular testing has been proposed for use at different stages of pregnancy depending on the presence of maternal symptoms. Thus, symptomatic patients might have had ZIKV infection diagnosed earlier than asymptomatic patients for whom a molecular diagnosis was proposed in cases of fetal anomalies, amniocentesis , or both, occurring later in pregnancy. Among infected pregnant women without prolonged viremia, 7 were asymptomatic with a continuous exposure and did not have molecular testing before amniocentesis, preventing accurate identification of the time of infection during pregnancy. Thus, we cannot exclude that some of these patients were in fact infected earlier in pregnancy and had an undetected prolonged viremia resulting in an exposure misclassification. This bias would result in an underestimation of the consequences of prolonged viremia because a case that included neurologic anomalies (CZS) in the reference group from infected mothers without prolonged viremia could in fact be related to prolonged viremia. Similarly, we cannot exclude a potential selection bias given that some patients were tested by RT-PCR because their fetus had anomalies at inclusion. Because this proportion did not differ between the groups (3/14 vs. 4/19), even if we consider only anomalies suggestive of fetal infection at inclusion (2/14 vs. 2/19), we would not expect relative risks to be significantly affected. However, this potential selection bias could overestimate absolute frequencies of fetal anomalies in RT-PCR–positive patients, and this bias seems to be inherent in contemporary cohorts because inclusion after the observation of fetal anomalies potentially related to Zika were common.>3 different samples tested). Our results are congruent with Meaney et al.  and the CDC report (25) also described fetuses without congenital infection or adverse outcomes from mothers with prolonged viremia , reflecting that other etiologies for adverse perinatal outcomes remain present even in the context of a ZIKV epidemic (In conclusion, prolonged maternal ZIKV viremia could be a marker for an increased risk for maternal–fetal transmission and subsequent adverse perinatal outcomes. Even if prolonged maternal viremia is not consistently present in cases of congenital infection, it might reflect active viral replication in the fetal–placental compartment and should lead to an enhanced prenatal and neonatal follow-up.Additional information on prolonged maternal Zika viremia as a marker of adverse perinatal outcomes.Additional figure for study of prolonged maternal Zika viremia as a marker of adverse perinatal outcomes.


Studies to date demonstrated the relatedness of mid‐upper arm circumference (MUAC) measurement of pregnant women to their anthropometry/weight. Hence, the objective was to determine whether maternal MUAC at different gestational age predicted birthweight, and if so, to identify which cut‐offs provided the best prediction of low birthweight (LBW) in pregnant women cohort. A total of 928 pregnant women, free of any obstetrical and medical complications known to affect fetal growth, were followed from 20 to 24 weeks' gestation till delivery. Weight, height, and MUAC were determined for the pregnant women, and gestational age along with newborns anthropometry was collected. The mean birthweight was 2.6 ± 0.460 kg. Maternal age, height, weight, MUAC (three time points), gestational age at delivery, and post‐natal weight showed positive correlation with birthweight, crown heel length, and head circumference of the neonates. The cut‐off limit with the best sensitivity–specificity  for MUAC was 23 cm, whereas maternal weight of 55 kg had sensitivity and specificity of 62.5 and 59.9 for predicting LBW. Maternal weight of 55 kg and MUAC value of 23 cm had almost similar sensitivity and specificity for predicting LBW. MUAC (≤23 cm) can be considered as a potential indicator of LBW where weighing of pregnant women is not feasible or when presentation for antenatal care is late, especially where pre‐pregnancy weights are not available. Though MUAC is identified as a marker to assess nutritional status of pregnant women by WHO, data on optimal cut‐off points are lacking especially in Indian context.This is the first study to demonstrate relationship between MUAC and maternal weight gain on three serial measurements.We found a similar sensitivity–specificity of MUAC for predicting LBW to that of maternal weight, suggesting the validation of MUAC.Contributing to validate cut‐off point of MUAC measurements to assess poor pregnancy outcomes, which can help in identifying women at risk.1Low birthweight (LBW) is recognized as an important determinant of neonatal mortality and morbidity, and it is one of the World Health Assembly targets by 2025. The annual average rate of reduction in LBW prevalence globally was only 1.2% between the year 2000 and 2015 . To affect the future survival of the newborn and to improve quality of adult life, neonatal birthweight (BW) should be improved, which in turn is directly dependent on maternal anthropometry , height (Ht), mid‐upper arm circumference (MUAC), and maternal body mass index (BMI) in the first trimester are suggested as good predictors of LBW. Nevertheless, pre‐pregnancy body mass index and gestational weight gain are the preferred anthropometric indicators to identify women at risk of producing LBW babies. However, in India, pregnant women begin to visit antenatal clinics after 10 to 12 weeks of pregnancy, and therefore, pre‐pregnancy weight may not be available to calculate BMI. Considering various influencing factors, studies have demonstrated that MUAC is closely related to maternal weight, and therefore, it has been suggested as an effective tool for maternal nutrition status screening  with 80% power the sample size required was 556. However, expecting attrition of 35% due to long follow‐up, the sample size was calculated to 751. But 928 were found to be eligible and were recruited for screening  pregnant women who fulfilled inclusion criteria were registered after obtaining written informed consent and were followed through during pregnancy till child birth. Of the 928, 615 and 563 turned up for follow‐up during 30–34 weeks' and >36 weeks' gestation, respectively; but delivery data such as BW and gestational age were collected from 804 of 928 recruited women. Only singleton deliveries were included in the final analysis of the data . MUAC was measured in the right arm at the level, midway between acromion and olecranon processes in centimetre, to the nearest decimal place. Triceps, biceps, and subscapular skinfold thickness were measured by trained nutritionists using a Lange skinfold caliper (nearest to 1 mm) according to standardized methods. The average of three measurements was recorded at each site. Maternal body composition was evaluated for all the three times during gestation. Post‐natal weight was collected after 24 hr but within 5 days after delivery. Two dedicated project staff, nutritionists were doing the anthropometry after due training and inter/intra rater reliability was done every 3 months to keep the CV below 10%. All the demographic and pregnancy details along with 24‐hr diet recall were collected from the participants.The babies were examined within 24 hr of delivery, and BWs, crown heel length, and head circumference were recorded using the Seca weighing scales (to the nearest 1 g), infanto‐metre, and measuring tape (to the nearest 1 cm), respectively. As per the WHO (1995) definition, newborns weighing less than 2.5 kg were considered as LBW neonates. Gestational age was determined by dating the last menstrual period and at the time of recruitment and was corrected by first trimester ultrasonographic findings if the difference exceeded 5 days. SGA neonates were determined by comparing the BW with fetal growth standard by gestation week. Neonates with BW less than 10th percentile of the standard population for gestation week were considered as small for gestational age  were calculated for predicting BW outcome for various MUAC cut‐offs. Pearson's correlation coefficients between longitudinal measurements of mothers' anthropometry and newborns' anthropometry were done. Significance considered at P value .05.Data were analysed using SPSS 17.0 for windows IBM, Chicago. Mean and 2.6Institutional ethical committee approval was taken bearing the number 04/I/2014.3SD gestational age at the time of recruitment was 23.6 ± 6.55. Of the 928 pregnant women followed, birth outcome data were collected from 804 pregnant women. Of the 804, 50.7% babies were males and the rest were females, and 73% delivered normally whereas 27% had caesarian section. The newborns were free of any congenital anomalies. The mean BW was 2.6 ± 0.460 kg. Frequency of LBW and preterm births (PTB) were 22.1% and 7.1 %, respectively, and 33% were SGA. One third (33%) of the babies were less than 10th percentile of the standard population  status, only 23.2 % had Hb concentration more than 11 g/dl. Although 55.3 % had mild anaemia (9 to 11 g/dl), 18.4 % had moderate anaemia (7 to 9 g/dl), and severe anaemia was prevalent in 3.2% of pregnant women at the time of recruitment.SD Wt was 48.6 ± 8.128 kg at the time of recruitment, which increased to 56.2 ± 8.788 kg by 38.5 ± 2.09 weeks of gestation. Over the same period, mean ± SD maternal MUAC increased from 23.7 ± 2.863 cm to 24.5 ± 2.866 cm. Predictably, mean age, height, weight, MUAC, and gestational age at delivery (GAD) were significantly low in LBW  are given in Table 3.1Table r = .202) and length (r = .138) but not with head circumference (r = −.001).Maternal weight and MUAC at all the three time points  were available for 485 mother neonate pairs. Table 3.2r = .890, P = .001; r = .861, P = .001; r = .844, P = .001). Furthermore, Pearson's correlation showed a significant association  between MUAC change and gestational weight gain during the course of pregnancy in this cohort. Similarly, there was strong association between MUAC changes and BW. Linear association between maternal MUAC at all three time points during pregnancy  with mean BW of the neonates is depicted in Figure As expected, Wt and MUAC had robust correlations at all the three time points of gestation  between MUAC changes and gestational weight gain during the 15‐week period of pregnancy in this cohort. Pregnant women with lower height, weight, MUAC, lower fat mass, and lean body mass had smaller babies. The best cut‐off limit with the highest sensitivity–specificity product for weight was 55 kg and height was 152 cm for LBW.MUAC cut‐off value of ≤23 cm measured during first, second, and third antenatal visits emerged as a good predictor of LBW and SGA neonates in this cohort. Maternal weight and height and as expected gestational age at delivery were related not only to BW but also to crown heel length and head circumference. This is the first study to demonstrate the relationship between MUAC and maternal weight gain on three serial measurements. Pearson's correlation showed association  that analysed maternal MUAC and LBW outcome, of which six studies showed positive association between MUAC and LBW, whereas two studies did not show any correlation . Maternal MUAC cut‐off of ≤23 cm from 20 weeks' gestation up till delivery can be considered for prediction of LBW. However, further studies need to be taken up with a nationally representative sample to validate this method.The authors declare that they have no conflicts of interest.RH designed the study concept and substantially contributed in executing and writing the manuscript. VD and RB handled the data collection. BV and RS substantially contributed in data analysis. RK, SS, and JJ involved in interpreting the results. RH and VD wrote the manuscript and all the authors reviewed and approved the final manuscript.Table S1. Maternal body composition at different stages of gestation with birth weight, length and HCClick here for additional data file.


HRP-2 RDTs ), for malaria were performed. Thick smears were prepared and Giemsa-stained as recommended; then slides were observed under microscopy and reported quantitatively; RDTs were reported qualitatively (positive or negative). Both RDTs and thick smear results were recorded on data collection sheet. This study included a total of 150 study participants, 87 (58%) females and 63 (42%) males. The patients included in the study did not receive any antimalarial drug. The mean age of the study participants was 31.6 ± 12.4 with the majority of participants being between 25 and 44 years and the minority being above 65 years. The sensitivity of RDT (HRP-2) was calculated and found to be 95.0%, whereas the sensitivity of Giemsa microscopy was 100%. The specificity of RDT (HRP-2) was calculated and found to be 59.2%, whereas the specificity of Giemsa microscopy was 100%. Negative and positive predictive values of RDT are 85.4% and 82.7%, respectively. Negative and positive predictive values of Giemsa microscopy were both 100%. According to the results of the current study, the sensitivity, specificity, and both positive and negative predictive values of Giemsa microscopy are higher than those of histidine-rich protein 2-based rapid diagnostic test for malaria. The results obtained in histidine-rich protein 2-based rapid diagnostic test for malaria parasites should be confirmed with tests with high specificity. Further studies should determine the most appropriate type of rapid diagnostic test of malaria diagnosis to be used in combination with Giemsa microscopy. In addition, sensitivity and specificity of RDT (HRP-2) and Giemsa microscopy should be assessed against molecular biology techniques.Malaria presents a diagnostic challenge in most tropical countries such as Rwanda. Microscopy remains the gold standard for diagnosing malaria, but it is labor intensive and depends upon the skill of the examiner. Malaria rapid diagnostic tests (RDTs) have been developed as an easy, convenient alternative to microscopy. This cross-sectional study was conducted at Rukara Health Center which is located in Eastern Province, Kayonza district, Rwanda. One hundred and fifty suspected cases of malaria, who attended Rukara Health Centre, during the period, from 21 Plasmodium falciparum  is the most pathogenic and is frequently fatal if untreated in time [Malaria is one of the highest killer diseases affecting most tropical countries, especially African countries. It affects over 500 million people worldwide and over one million children die annually from malaria . Of all  in time . Traditi in time . This olThe WHO makes the tentative recommendation that parasite-based diagnosis should be used in all suspected cases of malaria with the possible exception of children in high-prevalence areas and certain other situations . For thiRDTs are commercially available in kit forms with all necessary reagents and the ease of performance of the procedures does not require extensive training or equipments to perform or to interpret the results, and results are read in 12–15 min. RDTs mainly come in two forms. One is antigen-based and normally requires the use of haemolyzed red blood cells while the other is antibody-based and normally requires the use of extracted serum. Generally speaking, antibodies are better expressed in serum otherwise plasma could also stand in place of serum for the antibody-based method [Malaria presents a diagnostic challenge in most tropical countries including Rwanda. Microscopy remains the gold standard for diagnosing malaria, but it is labor-intensive and depends upon the skill of the examiner . MalariaTo determine the diagnostic performance between rapid malaria diagnostic test and microscopic-based stain techniques for diagnosis of malaria at Rukara Health Center.To determine the sensitivity of rapid malaria diagnostic test and microscopic-based stain techniques for diagnosis of malaria.To determine the specificity of rapid malaria diagnostic test and microscopic-based stain techniques for diagnosis of malaria.To determine the positive and negative predictive values of rapid malaria diagnostic test and microscopic-based stain techniques for diagnosis of malaria.st June to 30th July 2017. All patients who are not suspected of malaria diseases were excluded from this study.This study was conducted at Rukara Health Center. It is located at Kayonza district in Eastern Province, Rwanda. A cross-sectional study design was used in this study. Target population of this study are all suspected cases of malaria, from various sectors of Kayonza district, Eastern Province, Rwanda, who attended Rukara Health Center during the period from 21n = required sample size. z = confidence level 95% . p = estimated prevalence of malaria, we will take 11% obtained as the prevalence of malaria in Eastern Province . d = margin of error at 5 % .The sample size was estimated by usingSample size calculation is as follows:Finally, the sample size was 264 patients.A convenience sampling with consecutive design was used to select the research subjects of this study.In this study, the demographic data were collected from patient file to data collection form. These were filled with a study ID, demographic (gender and district), and malaria status on microscopy as well as RDTs. The collected data were checked for completeness, edited into Microsoft Excel 2010 sheet, and then imported into IBM SPSS for statistical analysis.μL blood = number of parasites counted x 8000 white blood cells/μL divided by number of white blood cells counted [Patient specimens (blood capillary) were used in RDTs for the diagnosis of malaria. Thick smears were prepared and Giemsa-stained as recommended . Giemsa-Data collection forms were used to collect data, and the information was inputted into a computer. The computer was used for safe storage and analysis of the data abstracted.Categorical measurements were reported as number and percentage. Quantitative measurements were reported as the mean ± SD (standard deviation). Sensitivity, specificity, positive predictive values, and negative predictive values of RDT in reference to the quantitative method were calculated by using the formulas given in Patients included in this manuscript did not receive any antimalarial drug before participating in the study.This study was revised and approved by a departmental Institutional Review Board committee in the school of Health Sciences of Mount Kenya University, Kigali. Ethical approval was also requested from research committee of the Rukara Health Center. To assure confidentiality, numbers were used as study ID instead of names or hospital ID on patient data collection forms.The demographic characteristics of the study subjects are given in Proportions of malaria by RDT and the quantitative method are given in Proportions of malaria by Giemsa microscopy and the quantitative method are given in The sensitivity of RDTs (HRP-2) and Giemsa microscopy in diagnosis of malaria is given in The specificity of RDTs (HRP-2) and Giemsa microscopy in diagnosis of malaria is given in Positive predictive value is the probability that subjects with a positive screening test truly have the disease. Negative predictive value is the probability that subjects with a negative screening test truly do not have the disease. Positive and negative predictive values of RDT (HRP-2) and Giemsa microscopy are calculated as given in This study showed the sensitivity of RDT (HRP-2) of 95.0% and the specificity of RDT (HRP-2) of 59.2%, whereas the specificity and sensitivity of Giemsa microscopy were 100%. Negative and positive predictive values of RDT were 85.4% and 82.7%, respectively. Negative and positive predictive values of Giemsa microscopy were 100% Tables –7. TheseSimilar studies were conducted across Africa, Nigeria , Angola Sensitivity of RDTs obtained in this study (95.0%) was too higher than that of the studies conducted by Olusola et al. in Nigeria (62.3%), Cláudia in Angola (60%), and Vincent Batwala et al. in Uganda (91.0%). The specificity of RDTs obtained in this study (59.2%) was lower than that of the studies conducted by Olusola et al. in Nigeria (87.4%) and Cláudia in Angola (94.3%). The NPV and PPV obtained in this study (85.4% and 82.7%) were similar to those obtained by Olusola et al. in Nigeria; however, different results were obtained in the study by Cláudia in Angola (94.8% and 70.7%) and Vincent Batwala et al. in Uganda (95.8% and 88%).The possible explanation of false-negative RDTs is deletions or mutations within the pfhrp-2 gene or by the prozone effect reported by others , 13. NevThere is a great impact of RDT and microscopy in treatment of malaria. In fact, if a patient is positive with RDT at the initial stage without any previous antimalarial drug history, the patient can be treated with antimalarial drugs. On the other hand, if RDT is negative at the initial stage, microscopy is needed in order to confirm the infection because it could be a deleterious mutation. It is also clear that if the quantity of parasites is very low, the false-negative result on microscopy could be due to lack of hands on expertise in microscope reading which is a common problem in capacity building. It is also worth noting that RDT detects genes, whereas microscopy detects parasites; therefore, if a patient revisits the health facility for almost similar symptoms, the RDT may be positive, whereas it can be negative on microscopy. On the other hand, this could be due to reemerging of the disease due to uncompleted dose. Therefore, it is recommended to redo both the tests before retreating the patient again in order to avoid any overdose.According to the results of the current study, sensitivity, specificity, and both positive and negative predictive values of Giemsa microscopy (All 100%) are higher than those of histidine-rich protein 2-based rapid diagnostic test for malaria (sensitivity (95%), specificity (59.2%), and PPV (82.7%) and NPV (85.4%). It is worth to say that RDT is an easy and rapid test for malaria diagnosis for quick intervention in treatment. The results obtained in histidine-rich protein 2-based rapid diagnostic test for malaria parasites should be confirmed with tests with high specificity. Further experimental studies should develop the most appropriate type of rapid diagnostic test of malaria diagnosis to be used in combination with Giemsa microscopy. In addition, the sensitivity and specificity of RDT (HRP-2) and Giemsa microscopy should be assessed against molecular biology techniques.


As Zambia continues to reduce its malaria incidence and target elimination in Southern Province, there is a need to identify factors that can reintroduce parasites and sustain malaria transmission. To examine the relative contributions of types of human mobility on malaria prevalence, this analysis quantifies the proportion of the population having recently traveled during both peak and nonpeak transmission seasons over the course of 2 years and assesses the relationship between short-term travel and malaria infection status. Among all residents targeted by mass drug administration in the Lake Kariba region of Southern Province, 602,620 rapid diagnostic tests and recent travel histories were collected during four campaign rounds occurring between December 2014 and February 2016. Rates of short-term travel in the previous 2 weeks fluctuated seasonally from 0.3% to 1.2%. Travel was significantly associated with prevalent malaria infection both seasonally and overall . The strength of association between travel and malaria infection varied by travelers’ origin and destination, with those recently traveling to high-prevalence areas from low-prevalence areas experiencing the highest odds of malaria infection (AOR: 7.38). Long-lasting insecticidal net usage while traveling was associated with a relative reduction in infections (AOR: 0.74) compared with travelers not using a net. Although travel was directly associated with only a small fraction of infections, importation of malaria via human movement may play an increasingly important role in this elimination setting as transmission rates continue to decline. Even still, continuous importation of parasites to areas of low reproductive capacity may perpetually fuel transmission even if the area is incapable of sustaining endemic transmission alone.18Among the risk factors for reintroducing malaria parasites into a population is the circulation of people to and from areas of ongoing transmission.24 Not all types of travel pose the same risk for infection nor does travel necessarily translate into a significant threat to low-prevalence areas. An individual’s duration and frequency of travel, risk while traveling, and risk management behaviors contribute to the probability of acquiring parasites.25 More broadly, the frequency of human movement, abundance of high-prevalence areas, and spatial connectedness of regional sources of transmission determine the frequency of higher risk travel and can result in differences in vulnerability between populations.26Within sub-Saharan Africa, previous studies have provided empirical evidence demonstrating a general association between travel and individual parasitemia, especially among individuals returning to lower prevalence areas.5 Following concentrated malaria control and elimination efforts, many of these health facility catchment areas (HFCAs) now see very low parasite prevalence, although areas of moderate prevalence (> 10% among children younger than 5 years) still remain.3 High vector control coverage and expansion of community case management have likely decreased receptivity in this region; however, current low infection prevalence, due in part to punctuated gains from mass treatment, does not likely reflect the capability of these areas to support higher transmission rates. Remaining areas of higher prevalence here may serve as nearby sources of importation, yet the extent to which people move between these areas, the impact routine travel has on the risk of infection, or the contribution of this movement to the remaining malaria transmission in lower prevalence areas is unclear. Efforts assessing the infection prevalence associated with various types of travel and quantifying population mobility within this region would provide insights into the role movement plays with respect to current elimination efforts. In addition, in the context of MDA, understanding the degree of mobility in this setting would supplement discussion of intervention impact, as higher rates of travel outside the study region would reduce the epidemiological coverage of treatments.Until recently, local transmission intensity in Southern Province has varied substantially between adjacent districts and their health facilities.Plasmodium falciparum malaria infection and identify characteristics of travel that modify that relationship, and 3) report the proportion of infections with a recent travel history. In addition, this analysis uses data from four time points over 2 years to examine seasonal differences in short-term travel rates and differences in the relationship between infection and travel, as malaria prevalence declined throughout the region.To this end, here we examine associations between human movement and malaria parasite prevalence in the Lake Kariba region, an area targeted for elimination in the next few years. Specifically, this analysis uses data collected during household visits during the trial to 1) quantify the proportion of the population that had recently traveled, 2) assess the relationship between recent travel and individual 5 In brief, the trial was conducted within 60 geographically delineated HFCAs covering approximately 15,800 km2 of the Lake Kariba region of Southern Province. Forty of the included HFCAs were randomly allocated (20 per arm) to receive one of two MDA treatment modalities. For each round of MDA conducted, field teams attempted to visit all households within these HFCAs.Four MDA rounds with dihydroartemisinin–piperaquine, coinciding with the low (two rounds) (June–December)- and high (two rounds) (January–May)-transmission seasons, were conducted each year for two consecutive years in Southern Province, Zambia, during December 2014, February 2015, October 2015, and February 2016 as part of a community randomized controlled trial. The aims and methods of the trial are discussed in detail elsewhere.P. falciparum malaria using histidine-rich protein 2 (HRP2) malaria rapid diagnostic tests (RDTs) , provided treatment as per the study protocol, and administered questionnaires on environmental and behavioral risk factors for malaria, including history of indoor residual spraying (IRS), long-lasting insecticide-treated net (LLIN) ownership and usage, and recent travel. Household members, or a representative, reported whether individuals had spent at least one evening away from their place of residence within the previous 2 weeks, including the trip destination, the total nights away, and whether during travel, an LLIN was used while sleeping. If a trip was within the study area of Southern Province, the destination HFCA was noted by the interviewer; otherwise, the destination district or country was reported.During household visits, all present household members aged 3 months or older were tested for P. falciparum prevalence (PfPR) measured as the proportion of RDT-positive children aged 1–59 months during a baseline cross-sectional survey in 2014, HFCAs were stratified into higher  or lower  transmission groups as part of the trial design . Adjusted odds ratios (AORs) were calculated controlling for participant age, gender, LLIN usage at home the previous evening, IRS at residence, trial arm, campaign round, and HFCA prevalence strata, including a random effect for the HFCA. Additional logistic models were used to estimate associations between travel and infection for each campaign round to assess differences in the relationship between infection and travel during various seasons, as parasite prevalence diminished throughout the study area with each subsequent round.All residents within HFCAs receiving either version of MDA—approximately 235,000 individuals in 37,000 households—were considered eligible for inclusion in this study. Consenting/assenting individuals visited during the trial with a valid RDT result and responding to questions on recent travel history were included in analysis. Data analysis was conducted in R version 3.5.1 .In total, 680,045 line listings of household members were produced during household visits across four mass treatment rounds, and 94.8% were present and consented to participate in malaria testing. Valid RDT results were recorded for 602,171 consenting participants (93.4%). Recent travel history was further available for 602,294 (99.7%) participants with a valid RDT result.2P < 0.001). The proportion of individuals traveling varied by HFCA but was generally greater in lower prevalence areas  than that in higher prevalence areas . Distributions of traveler characteristics were similar among residents from lower and higher prevalence areas except that the proportion of female travelers was slightly lower in higher prevalence HFCAs .Across the study area and all time points, 4,002 (0.7%) participants reported spending at least one evening away from home during the 2-week period before household visits. Compared with those reporting no travel, travelers were more likely older, male, to have slept under a net the previous evening at their place of residence, and to reside in a house that had not received IRS within the previous year . A smallP. falciparum infection by RDT was 5.2% across all rounds and HFCAs. Among those positive for malaria, 1.4% had reported recent travel. Infection prevalence was higher among travelers than those among non-travelers—11.0% versus 5.1% (χ2P < 0.001), respectively (P < 0.001) than those who had not traveled recently. The number of days spent traveling was also positively associated with infection. The magnitude of this association was even greater for those traveling for longer periods to higher prevalence areas. Stratifying by lower/higher HFCA prevalence, a greater proportion of prevalent infections from lower prevalence areas had a recent history of travel—4.0% versus 0.9% (χ2P < 0.001). Any travel was associated with increased odds of infection in both strata, although time spent away was not associated with an increase in the odds of infection among those from higher prevalence areas.The overall prevalence of ectively . AdjustiThroughout this population and across time points, a greater proportion of individuals reported travel to lower prevalence (33.3%) compared with higher prevalence areas 25.4%; . The remAnalysis stratified by traveler origin further parsed travel by the prevalence of both trip origin and destination and revealed differences in travel rates and malaria infection among subgroups . IndividRates of travel also differed by season and year , with hiAmong participants spending at least one evening away from home, 98.6% also reported on whether an LLIN was used while traveling. In total, 36.2% stated that a net had been used at least some evenings during the time away. Those sleeping under a net at home, aged between 5 and 15 years, and traveling from lower to higher prevalence areas were significantly more likely to use a net also while away. Net usage while traveling differed by season, reported at 45.4% and 44.3% during high-transmission seasons (rounds 2 and 4) compared with 33.7% and 32.9% during low-transmission seasons (rounds 1 and 3), respectively . MalariaP. falciparum infection status in a region of heterogeneous malaria transmission. Results demonstrated a clear association between recent overnight travel and malaria infection. Although malaria prevalence was estimated to be 5.1% over all MDA rounds, prevalence among those traveling within 2 weeks before testing was more than twice this amount. Malaria infections were even more likely among those taking trips of longer duration, especially to higher prevalence areas.Using survey data collected during four rounds of a mass treatment campaign spanning two consecutive years, we assessed recent short-term travel rates among residents and the relationship between this travel and individual The magnitude of the association between travel and infection differed by transmission levels at the origin and destination of travel, as well as seasonal differences in transmission. Among individuals from lower transmission areas, travel to areas of higher transmission was associated with a substantial increase in infections. Although travel was still associated with infection among individuals already living in higher transmission areas, destination did not seem to make a difference in the prevalence of infection likely because of the already higher probability of acquiring an infection within these areas. Seasonal differences in the travel–infection relationship corresponded to annual fluctuations in malaria transmission seen in this region .29 described seasonal differences in mobility among rural residents and reported that the rate of long-distance travel was noticeably reduced during rainy seasons. Here, we observed a similar pattern in travel, with a reduction in travel during high-transmission seasons believed to be due to changes in rural road conditions. Currently, reductions in travel likely serve to mitigate expected increases in the prevalence of infection attributable to travel during higher transmission seasons, but this effect may wane with future improvements in transportation infrastructure in rural areas.Only a small proportion of residents reported recent travel (from 0.3% to 1.2% across campaign rounds), resulting in relatively few prevalent infections with a recent history of travel throughout this population. Travel rates were slightly higher among those residing in lower prevalence areas possibly because of geographic differences between lower and higher prevalence catchment areas. Lower prevalence areas are closer to major thoroughfares connecting urban areas , whereas higher prevalence areas tend to be closer to Lake Kariba, where more challenging topography and poorer quality roads make travel more difficult. Observing human mobility within this same population, Searle et al.Throughout the 2-year period of this study, the season-specific proportion of infections related to travel increased with decreasing prevalence, suggesting that the proportional role of human movement in malaria infection may become more important as malaria prevalence further declines. Yet, widely implemented strategies for malaria control, such as LLIN distribution and promotion, may significantly reduce the role of regional movement in achieving and maintaining malaria-free areas. Net usage while traveling was associated with a significant reduction in the odds of malaria infection. Although overall net usage while traveling did not differ between residents of lower and higher transmission areas, higher rates were observed among subgroups traveling away from their area of residence, suggesting that differences in perceived risk may affect adoption of preventative measures. This is further supported by an increase in net usage while traveling during the peak transmission seasons of 2015 and 2016.It should be noted that the general limitations of cross-sectional designs used in other assessments of the impact of individual travel on malaria infection apply to this study as well. While estimating the strength of associations between travel and infection prevalence, the design of this study did not allow for further insight into the causal relationship between malaria and travel. Although travel has been recognized as a risk factor for malaria generally and in sub-Saharan Africa, this study did not allow us to identify the timing of infection with regard to travel—whether infection occurred before, during, or after travel, or whether travel was specifically to seek care for a symptomatic infection. The recall period used in this study was somewhat short at 2 weeks and likely led to both autochthonous infections being misclassified because of travel and vice versa. Infections acquired locally before leaving the area would have been considered as travel related in analysis, especially for very recent overnight trips. Infections acquired while traveling outside in the 2-week recall period would have been incorrectly classified as locally acquired. Extending the recall window would have addressed the latter issue but may have increased the probability of individuals seeking and receiving treatment before household visits, leading to some travel-related infections to appear negative by RDTs. Also, a longer recall period would have increased the probability of incorrectly classifying infections acquired after travel. It is likely that both types of misclassification occurred, although the resultant net change on effect estimates  is unclear.Single household visits provided snapshots of population travel rates but were limited in producing a comprehensive summary of circulation within this population needed to fully assess the role of movement in infections. Two-week travel rates do not indicate the frequency of travel or cumulative travel time throughout the year—a key dimension of the risk for parasite acquisition and thus importation. In addition, travel histories would have omitted those away at the time of surveys, underestimating the proportion of population traveling and, by extension, estimates of the proportion of infections attributable to travel. Timing of rounds may also have captured momentary peaks or troughs in travel rates not reflective of travel for the full season, although the duration of each round across the study area likely improved representativeness of these estimates.14 Nevertheless, their role may be significant to the long-term success of elimination efforts11 and should be included in a more robust assessment of the influence of human movement on continuing transmission.Finally, this study considered only travel among those residing in the study area. Presumably, individuals from other malaria-endemic provinces and countries travel to this area and may also contribute to local transmission. Unless visitors comprise the vast majority of travelers in the area, they likely contribute less to importation than returning residents.30 Previous studies on this topic have often been health facility based, using a case–control design to compare travel exposure and infection among patients.24 That design, although providing insights into the role of travel on individual malaria infection, does not lend itself to estimates of the relative importance of importation to current malaria prevalence and has often been limited in its ability to consider differences in risk due to characteristics of travel. Not all travel impose a substantial risk to malaria infection, and consideration of risk at trip destination is important; movement between areas with little transmission or during times with low vector activity is unlikely to have a substantial impact on travelers and onward transmission compared with travel to highly endemic areas.25 Including low-risk types of travel in estimations of the travel impact may understate the importance of some types of travel, indicating a need for better characterization of travel to adequately assess the impact of human movement and identify those most at risk, as previously suggested by Smith and Whittaker.25 Large-scale population-wide data collection used in this study provided a sufficient sample size to examine differences in categories of travel, such as travel direction, duration, distance, and season. Repeated data collection within the same population also provided insights into the changing role of travel in malaria infections during a period of diminishing prevalence.Conducting household surveys in the context of mass malaria testing afforded a rare opportunity for examining the prevalence of human movement and its association with malaria infection throughout a population. Questionnaires provided information on household and individual risk factors—including travel destination, IRS exposure, LLIN usage, age, etc.—for an estimated 80.7% of households among a population of approximately 235,000.1 and continued efforts of the national program and partners promise to push this even lower. Results from this analysis coincide with conclusions from others that short-term travel can pose a substantial risk of malaria infection and further suggest that this risk increases as local malaria prevalence declines. Although the proportion of infections directly associated with recent travel was relatively low, the threat of importation is likely to be increasingly important for achieving elimination regionally as prevalence declines. Moving forward, the national program will likely benefit from continued expansion of control and elimination efforts into surrounding provinces to address sources of transmission connected by movement. Last, the use of bed nets while traveling is promising, as it suggests that residents are risk conscious and receptive to adopting preventative measures even while outside their primary residence. Continued promotion of existing malaria prevention methods such as LLIN usage and/or targeting at-risk individuals with chemoprophylaxis may prove important for establishing and maintaining malaria-free areas in the long term.Southern Province, Zambia, has seen substantial reductions in overall malaria infection prevalence within the past few years,


The burden of malaria in Uganda remains high, but has become increasingly heterogenous following intensified malaria control. Travel within Uganda is recognized as a risk factor for malaria, but behaviours associated with travel are not well-understood. To address this knowledge gap, malaria-relevant behaviours of cohort participants were assessed during travel and at home in Uganda.vs at home were compared using log binomial regression models with generalized estimating equations adjusting for repeated measures in the same individual. Analysis of factors associated with LLIN adherence, such as destination and duration of travel, time to bed during travel, gender and age at time of travel, were assessed using log binomial regression models with generalized estimating equations adjusting for repeated measures in the same individual.Residents from 80 randomly selected households in Nagongera sub-county, Tororo district were enrolled into a cohort to study malaria in rural Uganda. All participants were given long-lasting insecticidal nets (LLINs) at enrolment and were evaluated every 4 weeks at the study clinic. Participants were asked if they had travelled overnight from their home, and if so, a questionnaire was administered to capture information on travel details and behaviours. Behaviour while travelling was assessed within 4 weeks following travel during the study clinic visit. Behaviour while at home was assessed using a similar questionnaire during two-weekly home visits. Behaviours while travelling Between October 2017 and October 2019, 527 participants were enrolled and assessed for travel. Of these, 123 (23.2%) reported taking 211 overnight trips; 149 (70.6%) trips were within Tororo. Participants were less likely to use LLINs when travelling than when at home ; this difference was noted for women  but not men . In an adjusted analysis, factors associated with LLIN use when travelling included destination  and duration of travel .Travellers, particularly women, were less likely to use LLINs when travelling than when at home. LLIN adherence was higher among those who travelled to non-IRS districts and for more than 1 week, suggesting that perceived malaria risk influences LLIN use. Strategies are needed to raise awareness of the importance of using LLINs while travelling. Malaria control interventions have been scaled-up globally, resulting in significant declines in malaria burden –3. DespiTravel is a well-recognized risk factor for malaria –18. StudAlthough travellers may be at increased risk of malaria due to exposure to higher malaria transmission , 21, chaThe study was conducted in Nagongera sub-county in Tororo district, Uganda, a research site for the Program for Resistance, Immunology, Surveillance, and Modelling for malaria in Uganda (PRISM). Details about the site and the two cohort studies led by PRISM (PRISM 1 from 2011 to 2017 and PRISM 2 from 2017 to 2019) have been described elsewhere . BrieflyThis study was nested in the PRISM 2 cohort study which has been described in detail elsewhere . BrieflyAll members of the enrolled households were screened and enrolled in the cohort study if they met the following selection criteria: (1) full-time resident of the selected household, (2) agreement to come to the study clinic for any illness and scheduled follow up, and (3) provision of written informed consent. Participants were followed up for 2 years, and the cohort was dynamic; any residents that were born into or joined the household were screened for enrolment during the course of the study. Participants were withdrawn from the study if they met the following criteria: (1) permanent movement out of Nagongera sub-county, (2) unable to be located for > 120 days, (3) withdrawal of informed consent, or (4) unable to comply with the study schedule and procedures. All enrolled participants were given LLINs at enrolment and were encouraged to come to the study clinic for all of their medical care.Participants were seen at the study clinic monthly for routine follow up. At these visits, participants were asked whether they had travelled overnight since the last visit. A detailed questionnaire was administered to those who travelled to capture data on destination and duration of travel, behavioural factors such as time to bed, and use of LLINs during travel. For malaria prevention, data were collected by asking the following question, “What measures did you take to prevent malaria?”. Responses included; none, slept under LLIN, used mosquito repellents, used mosquito coils, and took anti-malarial. Every 2 weeks, participants were visited at home and the same questionnaire was administered to collect data on behavioural factors while at home.Data were collected by trained study staff using standardized case record forms and double-entered using Microsoft Access . All the analyses were performed using Stata, version 14 . This analysis included data on any overnight travel and behavioural factors collected between October 2017 and October 2019. Overnight travel was defined as travel out of the sub-county of residence and spending at least one night away.p value < 0.05 was considered statistically significant.Behavioural factors including adherence to reported LLIN usage the prior night and time to bed were evaluated when study participants were at home and during overnight travel. LLINs use during travel was dichotomized into use most of the time during travel and no use, and time to bed was dichotomized into going to bed before 9 p.m. most of the time and going to bed at 9 p.m. or later mostly. Each overnight trip was paired with the most recent assessment at home as a comparison. For each pair, comparisons between behavioural factors during travel and while at home were made using log binomial regression models with generalized estimating equations adjusting for repeated measures in the same individual, and estimates were reported as relative risks (RR). In addition, an analysis of factors associated with LLIN adherence, such as destination and duration of travel, time to bed during travel, gender and age at time of travel, were also assessed using generalized estimating equations and expressed as relative risks. A From October 2017 through October 2019, all 531 eligible residents from 80 randomly selected households were enrolled; there were no exclusions  0.73, 95% CI 0.60–0.89, p = 0.002)   approach should be implemented to help educate people on malaria prevention and proper use of LLINs. Travellers should be encouraged to carry an extra LLIN when travelling, especially when visiting rural areas or those without ongoing IRS. Further research on innovative approaches to prevent malaria in travellers including portable LLINs, effective ways to influence behaviour and increase LLIN use, and acceptability of other malaria prevention measures such as mosquito repellents and chemoprophylaxis, should be encouraged.


P. falciparum endemic areas are at risk of developing malaria during pregnancy every year. Placental malaria is characterized by massive accumulation of infected erythrocytes in the intervillous space of the placenta, accompanied by infiltration of immune cells, particularly monocytes. The consequent local inflammation and the obstruction of the maternofetal exchanges can lead to severe clinical outcomes for both mother and child. Even if protection against the disease can gradually be acquired following successive pregnancies, the malaria parasite has developed a large panel of evasion mechanisms to escape from host defense mechanisms and manipulate the immune system to its advantage. Infected erythrocytes isolated from placentas of women suffering from placental malaria present a unique phenotype and express the pregnancy-specific variant VAR2CSA of the Plasmodium falciparum Erythrocyte Membrane Protein (PfEMP1) family at their surface. The polymorphic VAR2CSA protein is able to mediate the interaction of infected erythrocytes with a variety of host cells including placental syncytiotrophoblasts and leukocytes but also with components of the immune system such as non-specific IgM. This review summarizes the described VAR2CSA-mediated host defense evasion mechanisms employed by the parasite during placental malaria to ensure its survival and persistence.Over 30 million women living in  Plasmodium in sub-Saharan Africa, where P. falciparum is the most prevalent parasite species, accounting for 99.7% of estimated malaria cases , a condition that could cause very serious clinical outcomes for both mother and child, including maternal anemia  VAR2CSA has been identified as the sole parasite-derived protein interacting with placental CSA , which are interspaced by four inter-domain regions (IDs) –40 as weP. falciparum has therefore developed an efficient host defense evasion mechanism, which relies on a tight interaction between IEs and the syncytiotrophoblastic lining delimiting the intervillous spaces of the maternal portion of the placenta. As CSPGs are also present within the micro-vascular system, notably in the lungs and brain   . The decP1-null) . AlthougInterestingly, a study performed in an area of low prevalence of malaria, revealed gravidity-dependent differences in the capacity of peripheral blood mononuclear cells (PBMCs) to produce cytokines and chemokines in response to pregnancy-specific IEs . DespitePM induces VAR2CSA-specific immunoglobulin Gs (IgGs) belonging to the IgG1 subclass, and to a lower extent the IgG3 subclass , 88, botThe PfEMP1 binding sites on IgM have been located within the μ region of the fragment crystallizable (Fcμ) of polymeric immunoglobulins , and morP. falciparum genomes sequenced to date reveal the presence of one or more var2csa gene copies  between the DBL2x and DBL3x domains. A recent study has identified atypical extended or truncated VAR2CSA structures  (All the e copies –114. VARructures . Extendeructures . Within ructures . Var2csa members . One of ted ID2) .var2csa have been reported in different studies (var2csa (NTS-DBL5ε) from various strains and field isolates  were identified and the authors found an association between the 3D7-like clade and low birth-weight , followed by East Africa (27.5%), South East Asia (23.5%), and South America (21.1%). However, clade 3 is exclusively found in African parasite populations but appears to represent less than 1% of the var2csa sequences.Global sequence diversity and analysis of  studies –121 and isolates . Althougisolates , this laisolates , it possisolates . In a poh-weight . Only foh-weight . Indeed,var2csa genes within the parasite genome may also confer a greater capacity for antigenic variation and evasion of variant-specific immune responses   and FCR3 (clade 2) strains respectively, have been assessed in Phase I clinical trials in Europe and Africa  –134. Thein (CSP) , or viruin (CSP) –139.P. falciparum escapes host defenses, modulates the immune system and on how natural immunity develops during PM despite VAR2CSA polymorphisms is therefore crucial to design efficient and effective immuno-therapeutic approaches but also to appropriately evaluate them.Improving our understanding on how AT, J-PS, BG, and AC wrote the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by a grant from the ANR-18-IDEX-0001, IdEx Université de Paris attributed to AT and BG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest


Plasmodium falciparum histidine-rich proteins 2 (PfHRP2)-based rapid diagnostic tests (RDTs) are recommended for the diagnosis of falciparum malaria, particularly in endemic regions. However, genetic variability of the pfhrp2 gene threatens the usefulness of the test due to its impact on RDT sensitivity. This study aimed to investigate the diversity of pfhrp2 in malaria cases among children in Ghana.In the absence of microscopy, A cross-sectional study was conducted at the Adidome Government Hospital in the Volta Region of Ghana. A total of 50 children with mean age of 6.6 ± 3.5 years and diagnosed falciparum malaria were included. Blood samples were collected for complete blood count, malaria parasite identification and counting using auto analyzer and microscopy, respectively. DNA was isolated from blood-spotted Whatman filters, amplified and sequenced. Nucleotide sequences were translated in silico to corresponding amino acids and the deduced amino acids sequences were analyzed for diversity using Mega X.The number of repeats and number of each repeat within PfHRP2 varied between isolates. Twelve rare PfHRP2 repeat types, two of which are previously unreported, were identified in this study. The HRP2 sequence obtained in this study shared high similarities with isolates from Kenya. Using Baker’s regression model, Group B was the highest occurring type (58.0%). Screening of all sequences for epitopes recognized by PfHRP2-specific monoclonal antibodies (mAbs), the predominant motif was AHHAADAHH, which is recognized by the C1-13 mAbs.P. falciparum HRP2 in samples from Ghanaian children with symptomatic malaria. The findings of this study highlight the existence of extra amino acid repeat types which adds to the PfHRP2 antigenic variability.This study reports diversity of  Malaria, which causes substantial morbidity and mortality, is a major public health problem in sub-Saharan Africa, Asia and Latin America . It claiAlthough microscopic examination of stained blood smears remains the gold standard for malaria diagnosis, its benefit is limited by the absence of adequate skilled personnel and infrastructural and logistic resources, particularly in resource-poor areas . To overPlasmodium falciparum histidine-rich protein 2 (PfHRP2) for the detection of P. falciparum [Since its development in the 1990s, and with more than 200 currently available brands, there have been consistent reports of observable increase in malaria RDT sales worldwide . Most malciparum . The prelciparum , 13.pfhrp2 gene and extensive antigen diversity have contributed to discrepancies in RDT sensitivity [pfhrp2 gene deletion in 33 and 36% of microscopically-confirmed and PCR-confirmed RDT positive samples, respectively [P. falciparum isolates with deleted pfhrp2, and isolates with high pfhrp2 diversity [pfhrp2 gene deletion and diversity of the pfhrp2 gene in Ghana, a malaria-endemic country, are lacking.In 2010, Ghana implemented the test-before-treat guideline for malaria where RDT use was promoted to facilitate diagnosis . Howeversitivity –20, threectively . Over thiversity , 22–26, This study aimed to investigate the diversity of PfHRP2 in malaria cases among children in Ghana.P. falciparum. The regional prevalence of falciparum malaria is 45.27% among children [A cross-sectional study was conducted between January and June 2019 at the Adidome Government Hospital in the Volta Region of Ghana. The Volta Region has perennial malaria transmission, with the predominant parasite being Three ml of blood was aseptically obtained from each participant and dispensed into K3 EDTA tubes. Complete blood count was performed on the anticoagulated whole blood using Sysmex KX-21 N auto analyzer . To reassess falciparum malaria, thick and thin blood films were prepared and stained with 10% Giemsa for microscopic identification and counting of parasites. The parasite density was calculated by assuming a standard white blood cell (WBC) count of 8000/μl or 4.5 million red blood cells (RBCs)/μl in accordance with WHO standards .Additionally, 5 drops of the blood (each drop corresponding to about 30 μl of blood) were spotted onto Whatman™ filter papers , air dried and individually kept in zip-lock plastic bag for subsequent PCR analysis.pfhrp2 gene was performed using the semi-nested amplification approach, as previously described by Baker et al. [pfhrp2 gene are shown in Additional file P. falciparum and nuclease-free water were used as positive and negative controls, respectively. After secondary amplification, amplicons were separated by electrophoresis on 2% agarose gels, stained with ethidium bromide and visualized under UV light. Sequencing of the pfhrp2 gene was performed by Inqaba Biotechnical Industries (Pty) Ltd, South Africa (https://www.inqababiotec.co.za/). Nucleic acid sequences were deposited at the National Center for Biotechnology Information (NCBI) (Genbank accession numbers: MT094447-79).DNA isolation from Whatman filter papers was based on the Chelex-based technique as previously described . PCR ampr et al. . PCR reaMega X version 10.1.6  was usedvs 44.0%). The average RBC count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, WBC count, platelet count, and parasite density were 4.0 ± 0.7 × 106/µL, 10.5 ± 1.6 g/dL, 30.8 ± 4.6%, 77.2 ± 6.4 fL, 26.7 ± 4.1 pg, 34.5 ± 3.7 g/dL, 7.5 ± 2.7 × 103/µL, 101.0 (63.0–171.3) × 103/µL, and 35,411.0 /µL, respectively  and 7 (AHHAAH) had the highest frequency  and all PfHRP2 sequences terminated with type 12 (100.0%)  were obtained , which have been reported to be associated with RDT non-sensitivity  obtainedP. falciparum is the most common malaria-causing parasite. Microscopy is the gold standard for the diagnosis of malaria; however, in its absence, RDT has been recommended by WHO for use in malaria diagnosis. The majority of commercially available RDTs target the PfHRP2; however, the future benefit of these RDTs is in jeopardy due to reports of P. falciparum isolates that lack the pfhrp2 gene (deletion) and the presence of isolates with variants of the gene (diversity) [pfhrp2, especially in different parts of Africa where the disease exerts a high rate of morbidity and mortality, particularly among children.In Africa, versity) , 22, 23.pfhrp2 among P. falciparum isolates from children in Ghana. In contrast with a study by Amoah et al. in Ghana, who reported pfhrp2 gene deletion in 33 and 36% of microscopically confirmed and PCR-confirmed RDT-positive samples, respectively [pfhrp2 gene, indicating absence of deletion. This finding is in harmony with studies by Baker et al. [pfhrp2 gene deletion in this study (conducted in the middle belt) suggests that geographical differences could play a role in pfhrp2 gene diversity. Studies in other regions of the country are thus warranted. It should be noted, however, that inclusion of only symptomatic malaria cases and the limited sample size may have influenced the findings of this study. A larger sample size is recommended in future studies.This study presents an analysis of the diversity of ectively , all ther et al. , 20 and r et al. . Comparepfhrp2 genes into amino acid sequences, the isolates from this study shared some characteristics with previous published data. Amino acid repeat types 2, 6, 7 and 8 were found in all isolates whereas types 9 and 11 were absent. Few of the isolates had the type 4 repeat and only 10% had the type 13. All other repeat types were found in over 90% of the isolates. These findings are consistent with previous reports from other countries [After in silico translation of the ountries , 33–35.PDAHHVAD and AHHAAAHDEAALI) were identified in addition to 10 of the recently reported novel repeats which occurred at low frequencies. Together with previous reports, the findings of this study support the presence of yet-to-be defined repeat types and highlights that these novel types occur at relatively lower frequencies.The structural organization of the amino acid repeat types found in this study was highly diverse. Out of the 33 unqiue PfHRP2 sequences obtained, only a third occurred more than once. However, some repeat organizations were shared between isolates. Most of the sequences started with type 1 and all terminated with type 12 as is consistent with previous studies –20, 31. PfHRP2 diversity has been reported to influence the diagnosis of malaria using PfHRP2-based RDTs. In 2005, Baker et al. demonstrated, using logistic regression model, that the product of repeat types 2 and 7 affect inter-study sensitivity variation of PfHRP2-based RDTs, especially for samples with parasite densities ≤ 250 parasites/μl . In 2010pfhrp2 gene could potentially impact RDT sensitivity, the product of the pfhrp3 gene can compensate for its absence in relation to the sensitivity of the tests. Moreover, RDTs recommended by WHO are capable of detecting the PfHRP2/PfHRP3 antigens together with parasite-specific lactate dehydrogenase (pLDH) or Plasmodium aldolase [It is worthy of note that, although deletion/diversity of aldolase . Thus, tP. falciparum histidine-rich proteins 2 in samples from Ghanaian children with symptomatic malaria. Additionally, the findings of this study highlight the existence of extra amino acid repeat types which adds to the PfHRP2 antigenic variability. The findings of this study will contribute to the understanding of the performance of PfHRP2-based RDTs in the Ghanaian setting.This study reports diversity of Additional file 1. Primers used, BLASTP hits for HRP2 sequence modelled from Ghanaian isolates and BLASTP of Ghanaian HRP2 sequences for all 50 samples.