Cellulose filtration of blood from malaria patients for improving <i>ex vivo</i> growth of <i>Plasmodium falciparum</i> parasites

BACKGROUND: Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample. METHODS: In this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates from Tanzanian children admitted to hospital was tested. The parasites were allowed to expand in culture without subcultivation until 5 days after admission or the appearance of dead parasites and parasitaemia was determined daily. To investigate whether the filtration had an effect on clonality, P. falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture. RESULTS: The cellulose-filtered parasites grew to higher parasitaemia faster than non-filtered parasites seemingly due to a higher development ratio of ring stage parasites progressing into the late stages. Cellulose filtration had no apparent effect on clonality or var gene expression; however, evident differences were observed after only 4 days of culture in both the number of clones and transcript levels of var genes compared to the time of admission. CONCLUSIONS: Cellulose column filtration of parasitized blood is a cheap, applicable method for improving cultivation of P. falciparum field isolates for ex vivo based assays; however, when assessing phenotype and genotype of cultured parasites, in general, assumed to represent the in vivo infection, caution is advised. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12936-017-1714-2) contains supplementary material, which is available to authorized users

Haplotypes of the Endothelial Protein C Receptor (EPCR) Gene are Not Associated with Severe Malaria in Tanzania.

Endothelial protein C receptor (EPCR) was recently identified as a key receptor for Plasmodium falciparum erythrocyte membrane protein 1 mediating sequestration of P. falciparum-infected erythrocytes in patients suffering from severe malaria. Soluble EPCR (sEPCR) inhibits binding of P. falciparum to EPCR in vitro and increased levels of sEPCR have been associated with the H3 haplotype of the EPCR encoding PROCR gene. It has been hypothesized that elevated sEPCR levels, possibly linked to the PROCR H3 genetic variant, may confer protection against severe forms of malaria. This study determined the frequencies of PROCR haplotypes H1-4 and plasma levels of sEPCR in a Tanzanian study population to investigate a possible association with severe malaria. Study participants were children under 5 years of age admitted at the Korogwe District Hospital (N = 143), and diagnosed as having severe malaria (N = 52; including cerebral malaria N = 17), uncomplicated malaria (N = 24), or an infection other than malaria (N = 67). In addition, blood samples from 71 children living in nearby villages were included. The SNPs defining the haplotypes of PROCR gene were determined by post-PCR ligation detection reaction-fluorescent microsphere assay. Individuals carrying at least one H3 allele had significantly higher levels of sEPCR than individuals with no H3 alleles (P < 0.001). No difference in the frequency of H3 was found between the non-malaria patients, malaria patients or the village population (P > 0.1). Plasma levels of sEPCR differed between these three groups, with higher sEPCR levels in the village population compared to the hospitalized patients (P < 0.001) and higher levels in malaria patients compared to non-malaria patients (P = 0.001). However, no differences were found in the distribution of H3 (P = 0.2) or levels of sEPCR (P = 0.8) between patients diagnosed with severe and uncomplicated malaria. Frequencies of SNPs determining PROCR haplotypes were in concordance with other African studies. The PROCR H3 allele was associated with higher levels of sEPCR, confirming earlier findings, however, in this Tanzanian population; neither PROCR haplotype nor level of sEPCR was associated with severe malaria, however, larger studies are needed to confirm these findings

Plasma Ang2 and ADAM17 levels are elevated during clinical malaria; Ang2 level correlates with severity and expression of EPCR-binding PfEMP1

The pathogenesis of Plasmodium falciparum malaria involves a complex interplay between parasite adhesion and inflammatory response that includes release of cytokines and activation of the endothelium with accompanying release of Angiopoitin 2 (Ang2) to the plasma. A-disintegrin and metalloproteinase 17 (ADAM17) is a protein responsible for releasing cytokines, including Tumor Necrosis Factor α (TNFα), and shedding of adhesion proteins. In this study, we show that plasma levels of ADAM17 are increased in Tanzanian children hospitalized with a malaria infection compared with asymptomatic children but similar to children hospitalized with other infectious diseases. The plasma levels of ADAM17 decreased during recovery after an acute malaria episode. Plasma levels of Ang2 were associated with markers of malaria severity and levels of var transcripts encoding P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) containing Cysteine Rich Inter Domain Region α1 (CIDRα1) domains predicted to bind Endothelial Protein C receptor (EPCR). ADAM17 levels were not associated with expression of var genes encoding different PfEMP1 types when controlling for age. These data are the first to report ADAM17 plasma levels in malaria-exposed individuals, and support the notion that parasite sequestration mediated by EPCR-binding PfEMP1 is associated with endothelial activation and pathology in severe paediatric malaria

Mapping the Cord Blood Transcriptome of Pregnancies Affected by Early Maternal Anemia to Identify Signatures of Fetal Programming

Context Anemia during early pregnancy (EP) is common in developing countries and is associated with adverse health consequences for both mothers and children. Offspring of women with EP anemia often have low birth weight, which increases risk for cardiometabolic diseases, including type 2 diabetes (T2D), later in life. Objective We aimed to elucidate mechanisms underlying developmental programming of adult cardiometabolic disease, including epigenetic and transcriptional alterations potentially detectable in umbilical cord blood (UCB) at time of birth. Methods We leveraged global transcriptome- and accompanying epigenome-wide changes in 48 UCB from newborns of EP anemic Tanzanian mothers and 50 controls to identify differentially expressed genes (DEGs) in UCB exposed to maternal EP anemia. DEGs were assessed for association with neonatal anthropometry and cord insulin levels. These genes were further studied in expression data from human fetal pancreas and adult islets to understand their role in beta-cell development and/or function. Results The expression of 137 genes was altered in UCB of newborns exposed to maternal EP anemia. These putative signatures of fetal programming, which included the birth weight locus LCORL, were potentially mediated by epigenetic changes in 27 genes and associated with neonatal anthropometry. Among the DEGs were P2RX7, PIK3C2B, and NUMBL, which potentially influence beta-cell development. Insulin levels were lower in EP anemia-exposed UCB, supporting the notion of developmental programming of pancreatic beta-cell dysfunction and subsequently increased risk of T2D in offspring of mothers with EP anemia. Conclusions Our data provide proof-of-concept on distinct transcriptional and epigenetic changes detectable in UCB from newborns exposed to maternal EP anemia.Peer reviewe

Weight change during the first week of life and a new method for retrospective prediction of birthweight among exclusively breastfed newborns

Introduction: Identification of low birthweight and small for gestational age is pivotal in clinical management and many research studies, but in low‐income countries, birthweight is often unavailable within 24 h of birth. Newborn weights measured within days after birth and knowledge of the growth patterns in the first week of life can help estimate the weight at birth retrospectively. This study aimed to generate sex‐specific prediction maps and weight reference charts for the retrospective estimation of birthweight for exclusively breastfed newborns in a low‐resource setting. Material and methods: This was a prospective cohort study nested in a clinical trial of intermittent preventive treatment in pregnancy for malaria with either dihydroartemisinin–piperaquine with/without azithromycin or sulfadoxine–pyrimethamine in Korogwe District, north‐eastern Tanzania (Clinicaltrials.gov: NCT03208179). Newborns were weighed at birth or in the immediate hours after birth and then daily for 1 week. Reference charts, nadir, time to regain weight, and prediction maps were generated using nonlinear mixed‐effects models fitted to the longitudinal data, incorporating interindividual variation as random effects. Predictions and prediction standard deviations were computed using a linear approximation approach. Results: Between March and December 2019, 513 live newborns with birthweights measured within 24 h of delivery were weighed daily for 1 week. Complete datasets were available from 476 exclusively breastfed newborns. There was a rapid decline in weight shortly after delivery. The average weight loss, time of nadir, and time to regain weight were 4.3% (95% confidence interval [CI] 3.8–4.9) at 27 h (95% CI 24–30) and 105 h (95% CI 91–120) in boys and 4.9% (95% CI 4.2–5.6) at 28 h (95% CI 23–33) and 114 h (95% CI 93–136) in girls, respectively. The data were used to generate prediction maps with 1‐h time intervals and 0.05 kg weight increments showing the predicted birthweights and weight‐for‐age and weight‐change‐for‐age reference charts depicting variation in weight loss from 10%. Conclusions: The prediction maps and reference charts can be used by researchers in low‐resource settings to retrospectively estimate birthweights using weights collected up to 168 h after delivery, thereby maximizing data utilization. Clinical practitioners can also use the prediction maps to retrospectively classify newborns as low birthweight or small for gestational age

Reliability of Rapid Diagnostic Tests in Diagnosing Pregnancy-Associated Malaria in North-Eastern Tanzania.

Accurate diagnosis and prompt treatment of pregnancy-associated malaria (PAM) are key aspects in averting adverse pregnancy outcomes. Microscopy is the gold standard in malaria diagnosis, but it has limited detection and availability. When used appropriately, rapid diagnostic tests (RDTs) could be an ideal diagnostic complement to microscopy, due to their ease of use and adequate sensitivity in detecting even sub-microscopic infections. Polymerase chain reaction (PCR) is even more sensitive, but it is mainly used for research purposes. The accuracy and reliability of RDTs in diagnosing PAM was evaluated using microscopy and PCR. A cohort of pregnant women in north-eastern Tanzania was followed throughout pregnancy for detection of plasmodial infection using venous and placental blood samples evaluated by histidine rich protein 2 (HRP-2) and parasite lactate dehydrogenase (pLDH) based RDTs (Parascreen™) or HRP-2 only (Paracheck Pf® and ParaHIT®f), microscopy and nested Plasmodium species diagnostic PCR. From a cohort of 924 pregnant women who completed the follow up, complete RDT and microscopy data was available for 5,555 blood samples and of these 442 samples were analysed by PCR. Of the 5,555 blood samples, 49 ((proportion and 95% confidence interval) 0.9% [0.7 -1.1]) samples were positive by microscopy and 91 (1.6% [1.3-2.0]) by RDT. Forty-six (50.5% [40.5 - 60.6]) and 45 (49.5% [39.4 - 59.5]) of the RDT positive samples were positive and negative by microscopy, respectively, whereas nineteen (42.2% [29.0 - 56.7]) of the microscopy negative, but RDT positive, samples were positive by PCR. Three (0.05% [0.02 - 0.2]) samples were positive by microscopy but negative by RDT. 351 of the 5,461 samples negative by both RDT and microscopy were tested by PCR and found negative. There was no statistically significant difference between the performances of the different RDTs. Microscopy underestimated the real burden of malaria during pregnancy and RDTs performed better than microscopy in diagnosing PAM. In areas where intermittent preventive treatment during pregnancy may be abandoned due to low and decreasing malaria risk and instead replaced with active case management, screening with RDT is likely to identify most infections in pregnant women and out-performs microscopy as a diagnostic tool

Fetal growth and birth weight are independently reduced by malaria infection and curable sexually transmitted and reproductive tract infections in Kenya, Tanzania, and Malawi: A pregnancy cohort study

Objective Malaria and sexually transmitted and reproductive tract infections (STIs/RTIs) are highly prevalent in sub-Saharan Africa and associated with poor pregnancy outcomes. We investigated the individual and combined effects of malaria and curable STIs/RTIs on fetal growth in Kenya, Tanzania, and Malawi. Methods This study was nested within a randomized trial comparing monthly intermittent preventive treatment for malaria in pregnancy with sulfadoxine-pyrimethamine versus dihydroartemisinin-piperaquine, alone or combined with azithromycin. Fetal weight gain was assessed by serial prenatal ultrasound. Malaria was assessed monthly, and Treponema pallidum, Neisseria gonorrhoeae, Trichomonas vaginalis, Chlamydia trachomatis and bacterial vaginosis at enrolment and in the third trimester. The effect of malaria and STIs/RTIs on fetal weight/birthweight Z-scores was evaluated using mixed-effects linear regression. Results 1,435 pregnant women had fetal/birth weight assessed 3,950 times. Compared to women without malaria or STIs/RTIs (n=399), malaria-only (n=267), STIs/RTIs-only (n=410) or both (n=353) were associated with reduced fetal growth (adjusted mean difference in fetal/birth weight Z-score [95% CI]: malaria=-0.18 [-0.31,-0.04], p=0.01]; STIs/RTIs=-0.14 [-0.26,-0.03], p=0.01]; both=-0.20 [-0.33,-0.07], p=0.003). Paucigravidae experienced the greatest impact. Conclusion Malaria and STIs/RTIs are associated with poor fetal growth especially among paucigravidae women with dual infections. Integrated antenatal interventions are needed to reduce the burden of both malaria and STIs/RTIs

The choice of reference chart affects the strength of the association between malaria in pregnancy and small for gestational age: an individual participant data meta-analysis comparing the Intergrowth-21 with a Tanzanian birthweight chart

Background: The prevalence of small for gestational age (SGA) may vary depending on the chosen weight-for-gestational-age reference chart. An individual participant data meta-analysis was conducted to assess the implications of using a local reference (STOPPAM) instead of a universal reference (Intergrowth-21) on the association between malaria in pregnancy and SGA. Methods: Individual participant data of 6,236 newborns were pooled from seven conveniently identified studies conducted in Tanzania and Malawi from 2003–2018 with data on malaria in pregnancy, birthweight, and ultrasound estimated gestational age. Mixed-effects regression models were used to compare the association between malaria in pregnancy and SGA when using the STOPPAM and the Intergrowth-21 references, respectively. Results: The 10th percentile for birthweights-for-gestational age was lower for STOPPAM than for Intergrowth-21, leading to a prevalence of SGASTOPPAM of 14.2% and SGAIG21 of 18.0%, p < 0.001. The association between malaria in pregnancy and SGA was stronger for STOPPAM (adjusted odds ratio (aOR) 1.30 [1.09–1.56], p < 0.01) than for Intergrowth-21 (aOR 1.19 [1.00–1.40], p = 0.04), particularly among paucigravidae (SGASTOPPAM aOR 1.36 [1.09–1.71], p < 0.01 vs SGAIG21 aOR 1.21 [0.97–1.50], p = 0.08). Conclusions: The prevalence of SGA may be overestimated and the impact of malaria in pregnancy underestimated when using Intergrowth-21. Comparing local reference charts to global references when assessing and interpreting the impact of malaria in pregnancy may be appropriate

Comparative genomics revealed adaptive admixture in Cryptosporidium hominis in Africa

Cryptosporidiosis is a major cause of diarrhoeal illness among African children, and is associated with childhood mortality, malnutrition, cognitive development and growth retardation. Cryptosporidium hominis is the dominant pathogen in Africa, and genotyping at the glycoprotein 60 (gp60) gene has revealed a complex distribution of different subtypes across this continent. However, a comprehensive exploration of the metapopulation structure and evolution based on whole-genome data has yet to be performed. Here, we sequenced and analysed the genomes of 26 C. hominis isolates, representing different gp60 subtypes, collected at rural sites in Gabon, Ghana, Madagascar and Tanzania. Phylogenetic and cluster analyses based on single-nucleotide polymorphisms showed that isolates predominantly clustered by their country of origin, irrespective of their gp60 subtype. We found a significant isolation-by-distance signature that shows the importance of local transmission, but we also detected evidence of hybridization between isolates of different geographical regions. We identified 37 outlier genes with exceptionally high nucleotide diversity, and this group is significantly enriched for genes encoding extracellular proteins and signal peptides. Furthermore, these genes are found more often than expected in recombinant regions, and they show a distinct signature of positive or balancing selection. We conclude that: (1) the metapopulation structure of C. hominis can only be accurately captured by whole-genome analyses; (2) local anthroponotic transmission underpins the spread of this pathogen in Africa; (3) hybridization occurs between distinct geographical lineages; and (4) genetic introgression provides novel substrate for positive or balancing selection in genes involved in host–parasite coevolution