Plasmodium Falciparum DNA Research Articles

Optimized plasmid loading of human erythrocytes for Plasmodium falciparum DNA transfections

In vitro modification of Plasmodium falciparum genes is the cornerstone of basic and translational malaria research. Achieved through DNA transfection, these modifications may entail altering protein sequence or abundance. Such experiments are critical for defining the molecular mechanisms of key parasite phenotypes and for validation of drug and vaccine targets. Despite its importance, successful transfection remains difficult and is a resource-intensive, rate-limiting step in P. falciparum research. Here, we report that inefficient loading of plasmid into erythrocytes limits transfection efficacy with commonly used electroporation methods. As these methods also require expensive instrumentation and consumables that are not broadly available, we explored a simpler method based on plasmid loading through hypotonic lysis and resealing of erythrocytes. We used parasite expression of a sensitive NanoLuc reporter for rapid evaluation and optimization of each step. Hypotonic buffer composition, resealing buffer volume and composition, and subsequent incubation affected plasmid retention and successful transfection. While ATP was critical for erythrocyte resealing, addition of Ca++ or glutathione did not improve transfection efficiency, with increasing Ca++ concentrations proving detrimental to outcomes. Compared with either the standard electroporation method or a previously reported hypotonic loading protocol, the optimized method yields greater plasmid loading and higher expression of the NanoLuc reporter 48 h after transfection. It also produced significantly faster outgrowth of parasites in transfections utilizing either episomal expression or CRISPR-Cas9 mediated integration. This new method produces higher P. falciparum transfection efficiency, reduces resource requirements and should accelerate molecular studies of malaria drug and vaccine targets.

Comparison of Three Methods to Extract Plasmodium falciparum DNA from Whole Blood and Dried Blood Spots.

This study aimed to compare the effectiveness of three DNA extraction methods: the GF-1 Blood DNA Extraction Kit (GF-1 BD Kit), which employs a spin column along with lysing and washing buffers; the tris-ethylenediaminetetraacetic acid and proteinase K (TE-pK) method, which utilizes a combination of TE buffer and proteinase K for cell lysis; and DNAzol® Direct (DN 131), a single reagent combined with heating for the extraction process. Plasmodium falciparum DNA was extracted from both whole blood and dried blook spots (DBSs), with consideration of DNA concentration, purity, cost, time requirement, and limit of parasite detection (LOD) for each method. The target gene in this study was 18S rRNA, resulting in a 395-bp product using specific primers. In the comparative analysis, the DN 131 method yielded significantly higher DNA quantities from whole blood and DBSs than the GF-1 BD Kit and TE-pK methods. In addition, the DNA purity obtained from whole blood and DBSs using the GF-1 BD Kit significantly exceeded that obtained using the TE-pK and DN 131 methods. For LOD, the whole blood extracted using the DN 131, GF-1 BD Kit, and TE-pK methods revealed 0.012, 0.012, and 1.6 parasites/µL, respectively. In the case of DBSs, the LODs for the DN 131, GF-1 BD Kit, and TE-pK methods were 1.6, 8, and 200 parasites/µL, respectively. The results revealed that the TE-pK method was the most cost-effective, whereas the DN 131 method showed the simplest protocol. These findings offer alternative approaches for extracting Plasmodium DNA that are particularly well-suited for large-scale studies conducted in resource-limited settings.

Predominance of K1 Allele in Erythrocytic form of Plasmodium falciparum MSP-1 Gene Signifies Severe Malaria in Jos, Nigeria

Aim: The study explored the diversity of Plasmodium falciparum Merozoite Surface Protein 1 (msp-1) gene sequence and delineated the K1 allele occurrence and profile. The Plasmodium falciparum msp-1 gene encodes an antigen that is being proposed as a major vaccine candidate against the parasite infections.
 Study Design: The design of the study was experimental.
 Place and Duration of Study: The study was undertaken, between October 2018 and June 2019, in the Department of Biochemistry, Faculty of Basic Medical Sciences, College of Health Sciences, University of Jos, Nigeria.
 Methodology: The DNA of Plasmodium falciparum was extracted from 117 blood samples of malaria patients confirmed by microscopy in three different hospitals in Jos. The msp-1 (block 2) allelic family’s genotyping was carried out using PCR and nested PCR techniques. Sequencing and Bioinformatics of the K1 alleles were done to further identify the K1 alleles.
 Results: Out of the 117 DNA extracted, 13 samples were positive for Plasmodium falciparum and each was genotyped for msp-1. K1 was the most predominant allele (6/13) compared to MAD20 (4/13) and R033 (2/13). The allelic frequency of K1 was calculated to be 46.15%. The 225 bp Plasmodium falciparum K1 allele of the msp-1 gene studied displayed polymorphism. However, the sequence was very similar to those of Plasmodium falciparum already characterized.
 Conclusion: Predominance of K1 allele is an indication that Jos is endemic to severe Plasmodium falciparum malaria.

Detection of Plasmodium falciparum in Saliva and Stool Samples from Children Living in Franceville, a Highly Endemic Region of Gabon.

Due to the difficulty of obtaining blood samples, which is the invasive method that is currently used for the detection of Plasmodium spp., alternative diagnostic sampling methods that are effective and non-invasive are needed, particularly for long-term studies. Saliva and stool samples from malaria-infected individuals contain trace amounts of Plasmodium DNA and therefore could be used as alternatives. Malaria was screened using rapid diagnosis tests and confirmed via microscopy. Nested PCR tests targeting the Plasmodium falciparum-specific STEVOR gene were performed for blood, saliva and stool samples that were positive for malaria. Three hundred sixty-seven (367) children were enrolled and eighty (22.22%) were confirmed to be positive for malaria. Matched blood, saliva and stool samples were available for 35 children. By using blood smears as the gold standard for the diagnosis of malaria, our study indicates that Plasmodium DNA was more detectable in blood (100%) than in saliva (22.86%) and stools (14.29%). Applying qPCR to the STEVOR gene to detect Plasmodium falciparum DNA in saliva and stool samples cannot be considered as an alternative to the current malaria detection processes using blood specimens.

Codetection of Plasmodium falciparum in Children Hospitalized With Dengue Fever in the Dominican Republic.

Cases of malaria and dengue in the Dominican Republic both spiked in 2019, but their rates of codetection are poorly characterized, especially in children. We performed a prospective, observational study in January to December 2019 at the Hospital Infantil Robert Reid Cabral, in the Dominican Republic, enrolling hospitalized children with a clinical suspicion of dengue fever. Participants with a positive plasma dengue IgM antibodies were included in this study. Clinical and hospital data were abstracted, and dried blood spot samples were collected from participants and tested with quantitative polymerase chain reaction to detect the presence of Plasmodium falciparum DNA. A total of 429 children with serological evidence of acute dengue were included in this study, of whom 1.4% (n = 6/429) had codetection of dengue and malaria. There were no significant differences in fever duration or presence of vomiting, abdominal pain and rash between both groups. Children with dengue and malaria codetection were numerically more often admitted to the pediatric intensive care unit, despite no differences found in overall clinical severity. The codetection of malaria and dengue in children was overall uncommon in our Dominican Republic cohort despite the rise in cases in 2019 but may be associated with a more severe hospital course. Further epidemiological and cohort studies to characterize the risk of both pathogens as case numbers fluctuate will be important to better understand the dynamics of coinfections.

Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5–8 fold elevation in the mutation rate, with an increase of 13–28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an “irresistible” compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this “mutator” parasite can be leveraged to drive P. falciparum resistome discovery.

Genotyping of Anopheles mosquito blood meals reveals nonrandom human host selection: implications for human-to-mosquito Plasmodium falciparum transmission

BackgroundControl of malaria parasite transmission can be enhanced by understanding which human demographic groups serve as the infectious reservoirs. Because vector biting can be heterogeneous, some infected individuals may contribute more to human-to-mosquito transmission than others. Infection prevalence peaks in school-age children, but it is not known how often they are fed upon. Genotypic profiling of human blood permits identification of individual humans who were bitten. The present investigation used this method to estimate which human demographic groups were most responsible for transmitting malaria parasites to Anopheles mosquitoes. It was hypothesized that school-age children contribute more than other demographic groups to human-to-mosquito malaria transmission.MethodsIn a region of moderate-to-high malaria incidence in southeastern Malawi, randomly selected households were surveyed to collect human demographic information and blood samples. Blood-fed, female Anopheles mosquitoes were sampled indoors from the same houses. Genomic DNA from human blood samples and mosquito blood meals of human origin was genotyped using 24 microsatellite loci. The resultant genotypes were matched to identify which individual humans were sources of blood meals. In addition, Plasmodium falciparum DNA in mosquito abdomens was detected with polymerase chain reaction. The combined results were used to identify which humans were most frequently bitten, and the P. falciparum infection prevalence in mosquitoes that resulted from these blood meals.ResultsAnopheles females selected human hosts non-randomly and fed on more than one human in 9% of the blood meals. Few humans contributed most of the blood meals to the Anopheles vector population. Children ≤ 5 years old were under-represented in mosquito blood meals while older males (31–75 years old) were over-represented. However, the largest number of malaria-infected blood meals was from school age children (6–15 years old).ConclusionsThe results support the hypothesis that humans aged 6–15 years are the most important demographic group contributing to the transmission of P. falciparum to the Anopheles mosquito vectors. This conclusion suggests that malaria control and prevention programmes should enhance efforts targeting school-age children and males.

A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples

A fast, simple, easy, efficient, and inexpensive method for DNA extraction from malaria parasites collected on filter paper would be very useful for molecular surveillance. The quality and quantity of DNA are critical to molecular diagnosis and analysis. Here, we developed a simple alkali lysis method for DNA extraction from blood samples on filter paper. The results showed that 10–50 mM NaOH and deionized water all effectively isolated parasite DNA at higher parasitemia, as witnessed by successful PCR amplification, while at a parasitemia of 0.01%, the 10 mM NaOH lysis condition generated the best results. Furthermore, DNA extracted by this method was successfully used to amplify a fragment of > 2000 bp. This method successfully extracted DNA from 1 µl of blood at a parasitemia as low as 0.0001% (equivalent to 5 parasites /µl). The DNA isolated by the 10 mM NaOH lysis method was stable to yield PCR products after storage at 4 °C or − 20 °C for 12 months. These results indicate that this alkali lysis method is simple, effective, sensitive, and inexpensive for isolating stable Plasmodium DNA from dried blood spots on filter paper.

Genetic diversity of Plasmodium vivax isolates from pregnant women in the Western Brazilian Amazon: a prospective cohort study

Each year, 92 million pregnant women are at risk of contracting malaria during pregnancy, with the underestimation of the mortality and morbidity burden associated with Plasmodium vivax. During pregnancy, P.vivax infection is associated with low birth weight, maternal anaemia, premature delivery, and stillbirth. In the State of Acre (Brazil), high transmission leaves pregnant women at greater risk of contracting malaria and having a greater number of recurrences. The study of genetic diversity and the association of haplotypes with adverse pregnancy effects is of great importance for the control of the disease. Here we investigate the genetic diversity of P.vivax parasites infecting pregnant women across their pregnancies. P.vivax DNA was extracted from 330 samples from 177 women followed during pregnancy, collected in the State of Acre, Brazil. All samples were negative for Plasmodium falciparum DNA. Sequence data for the Pvmsp1 gene was analysed alongside data from six microsatellite (MS) markers. Allelic frequencies, haplotype frequencies, expected heterozygosity (HE) were calculated. Whole genome sequencing (WGS) was conducted on four samples from pregnant women and phylogenetic analysis performed with other samples from South American regions. Initially, the pregnant women were stratified into two groups-1 recurrence and 2 or more recurrences-in which no differences were observed in clinical gestational outcomes or in placental histological changes between the two groups. Then we evaluated the parasites genetically. An average of 18.5 distinct alleles were found at each of the MS loci, and the HE calculated for each marker indicates a high genetic diversity occurring within the population. There was a high percentage of polyclonal infections (61.7%, 108/175), and one haplotype (H1) occurred frequently (20%), with only 9 of the haplotypes appearing in more than one patient. Most pregnant women had polyclonal infections that could be the result of relapses and/or re-infections. The high percentage of H1 parasites, along with the low frequency of many other haplotypes are suggestive of a clonal expansion. Phylogenetic analysis shows that P.vivax population within pregnant women clustered with other Brazilian samples in the region. FAPESP and CNPq - Brazil.

High burden of malaria among Malawian adults on antiretroviral therapy after discontinuing prophylaxis.

Many individuals living with the human immunodeficiency virus (HIV) infection and receiving antiretroviral therapy (ART) reside in areas at high risk for malaria but how malaria affects clinical outcomes is not well described in this population. We evaluated the burden of malaria infection and clinical malaria, and impact on HIV viral load and CD4 + cell count among adults on ART. We recruited Malawian adults on ART who had an undetectable viral load and ≥250 CD4 + cells/μl to participate in this randomized trial to continue daily trimethoprim-sulfamethoxazole (TS), discontinue daily co-trimoxazole, or switch to weekly chloroquine (CQ). We defined clinical malaria as symptoms consistent with malaria and positive blood smear, and malaria infection as Plasmodium falciparum DNA detected from dried blood spots (collected every 4-12 weeks). CD4 + cell count and viral load were measured every 24 weeks. We used Poisson regression and survival analysis to compare the incidence of malaria infection and clinical malaria. Clinicaltrials.gov NCT01650558. Among 1499 participants enrolled, clinical malaria incidence was 21.4/100 person-years of observation (PYO), 2.4/100 PYO and 1.9/100 PYO in the no prophylaxis, TS, and CQ arms, respectively. We identified twelve cases of malaria that led to hospitalization and all individuals recovered. The preventive effect of staying on prophylaxis was approximately 90% compared to no prophylaxis (TS: incidence rate ratio [IRR] 0.11, 95% confidence interval [CI] 0.08, 0.15 and CQ: IRR 0.09, 95% CI 0.06, 0.13). P. falciparum infection prevalence among all visits was 187/1475 (12.7%), 48/1563 (3.1%), and 29/1561 (1.9%) in the no prophylaxis, TS, and CQ arms, respectively. Malaria infection and clinical malaria were not associated with changes in CD4 + cell count or viral load. In clinically stable adults living with HIV on ART, clinical malaria was common after chemoprophylaxis stopped. However, neither malaria infection nor clinical illness appeared to affect HIV disease progression.

G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum.

G-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen.

Properties of Plasmodium falciparum with a Deleted Apicoplast DNA Gyrase.

ABSTRACTMalaria parasites have three genomes: a nuclear genome, a mitochondrial genome, and an apicoplast genome. Since the apicoplast is a plastid organelle of prokaryotic origin and has no counterpart in the human host, it can be a source of novel targets for antimalarials. Plasmodium falciparum DNA gyrase (PfGyr) A and B subunits both have apicoplast-targeting signals. First, to test the predicted localization of this enzyme in the apicoplast and the breadth of its function at the subcellular level, nuclear-encoded PfGyrA was disrupted using CRISPR/Cas9 gene editing. Isopentenyl pyrophosphate (IPP) is known to rescue parasites from apicoplast inhibitors. Indeed, successful growth and characterization of PfΔGyrA was possible in the presence of IPP. PfGyrA disruption was accompanied by loss of plastid acyl-carrier protein (ACP) immunofluorescence and the plastid genome. Second, ciprofloxacin, an antibacterial gyrase inhibitor, has been used for malaria prophylaxis, but there is a need for a more detailed description of the mode of action of ciprofloxacin in malaria parasites. As predicted, PfΔGyrA clone supplemented with IPP was less sensitive to ciprofloxacin but not to the nuclear topoisomerase inhibitor etoposide. At high concentrations, however, ciprofloxacin continued to inhibit IPP-rescued PfΔGyrA, possibly suggesting that ciprofloxacin may have an additional nonapicoplast target in P. falciparum. Overall, we confirm that PfGyrA is an apicoplast enzyme in the malaria parasite, essential for blood-stage parasites, and a possible target of ciprofloxacin but perhaps not the only target.

Getting to zero: micro-foci of malaria in the Solomon Islands requires stratified control

BackgroundThe Solomon Islands has made significant progress in the control of malaria through vector control, access and use of improved diagnostics and therapeutic drugs. As transmission is reduced there is a need to understand variations in transmission risk at the provincial and village levels to stratify control methods.MethodsA cross-sectional survey of malaria in humans was conducted in the Solomon Islands during April 2018. Nineteen villages across 4 provinces were included. The presence of Plasmodium species parasites in blood samples was detected using PCR.ResultsBlood samples were analysed from 1,914 participants. The prevalence of DNA of Plasmodium falciparum was 1.2 % (n = 23) and for Plasmodium vivax was 1.5 % (n = 28). 22 % (n = 5/23) of P. falciparum DNA positive participants were febrile and 17 % of P. vivax DNA positive participants (n = 5/28). The prevalence of both P. falciparum and P. vivax was extremely spatially heterogeneous. For P. falciparum, in particular, only 2 small foci of transmission were identified among 19 villages. Plasmodium falciparum infections were uniformly distributed across age groups. Insecticide-treated bed net use the night prior to the survey was reported by 63 % of participants and significantly differed by province.ConclusionsMalaria transmission across the Solomon Islands has become increasingly fragmented, affecting fewer villages and provinces. The majority of infections were afebrile suggesting the need for strong active case detection with radical cure with primaquine for P. vivax. Village-level stratification of targeted interventions based on passive and active case detection data could support the progress towards a more cost-effective and successful elimination programme.

OMNIGEN KIT ACCURACY FOR PLASMODIUM FALCIPARU

Malaria is a serious public health burden in Senegal. Accurate diagnosis is essential to avoided unnecessary presumptive treatment. Malaria diagnosis currently relies on identifying malaria parasite using microscopy and detecting soluble parasite antigens by Rapid Diagnostic Tests (RDT). These techniques do not detect low-level, sub-patent malaria infections and are inherently hazardous and invasive. To overcome these obstacles, alternatives diagnostics method were explored. In contrast to blood, saliva presents a reduced biohazard and can be painlessly collected in relatively large quantities by individuals with moderate training. The objective of this study was to use saliva collected in OmnIGEN Kits as an alternative sample for malaria detection. Methods: A total of 77 febril patients tested malaria positive by mRDT were enrolled in this study. From each patient, blood sample was collected for dried blood spot and blood smear; and saliva sample on OMNIGEN kit. Parasite density was determined from smear and Plasmodium falciparum DNA was extracted from both dried blood spot and saliva samples collected from the same patients. Extracted DNA was amplied by qPCR machine. Results: Malaria prevalence using qPCR was 98,7% and 60% respectively with blood and saliva. Compared to blood, saliva showed a sensitivity; specicity; positive predictive value of 60%; 100 %; and 100 %. The concordance between parasites detection from saliva and blood was (p = 0.45). In addition, no difference was found between these two methods and the microscopy counting. Conclusions:Saliva could be a non-invasive alternative method for P. falciparum detection and epidemiological surveillance in country with limited ressources.

Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples.

Background:Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples.Methods:This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016–2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction).Results:The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant.Conclusion:We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.

Malaria during pregnancy and transplacental transfer of Kaposi sarcoma-associated herpesvirus (KSHV) antibodies: a cohort study of Kenyan mother and child pairs

BackgroundKaposi sarcoma-associated herpesvirus (KSHV) seroprevalence in sub-Saharan African children can range up to 50% by age 2 years but factors affecting early age of KSHV infection are not well understood. Malaria during pregnancy has been associated with hindered transplacental transfer of antibodies to several pathogens but whether it affects transplacental transfer of KSHV antibodies is unknown. We aimed to determine if in utero malaria exposure reduced the transfer of KSHV antibodies across the placenta.MethodsA cohort study in Kisumu, Kenya enrolled pregnant women at their first antenatal clinic (ANC) visit and followed them through delivery. We included 70 KSHV-positive, HIV-negative mothers and their children. KSHV antibody levels were measured by ELISA (K8.1, ORF73) and multiplex assay (K8.1, ORF73, K10.5, ORF38, ORF50). Transplacental transfer of antibodies was measured by the cord to maternal blood ratio (CMR) of KSHV antibodies. Malaria during pregnancy was defined as detection of Plasmodium falciparum (Pf) DNA at any ANC visit or delivery. Among women with malaria during pregnancy, we examined time of last malaria infection prior to delivery (< 27 vs. 27+ weeks gestation) and malaria incidence rate (MIR) (episodes/100 person-weeks).ResultsKSHV seroprevalence (positive for K8.1 or ORF73 by ELISA) among pregnant women was 88%. Neither malaria during pregnancy, malaria infection timing, nor MIR were associated with maternal delivery KSHV antibody blood levels. Maternal delivery and cord blood KSHV antibody levels were highly correlated but these correlations did not differ by malaria during pregnancy. KSHV transplacental antibody transfer was not associated with malaria during pregnancy, malaria infection timing, nor MIR.ConclusionsMalaria during pregnancy does not appear to affect transfer of KSHV antibodies across the placenta.

Factors associated with sub-microscopic placental malaria and its association with adverse pregnancy outcomes among HIV-negative women in Dar es Salaam, Tanzania: a cohort study

BackgroundMalaria infection during pregnancy has negative health consequences for both mothers and offspring. Sub-microscopic malaria infection during pregnancy is common in most African countries. We sought to identify factors associated with sub-microscopic placental malaria, and its association with adverse pregnancy outcomes among HIV-negative pregnant women in Dar es Salaam, Tanzania.MethodsWe recruited a cohort of pregnant women during their first trimester and assessed for the occurrence of placental malaria and pregnancy outcomes. The follow-up was done monthly from recruitment until delivery. Histopathology placental malaria positive results were defined as the presence of malaria pigment or parasitized erythrocytes on the slide (histology-positive (HP)), and the sub-microscopic placental infection was defined as positive Plasmodium falciparum DNA by polymerase chain reaction (DNA PCR) amplification in a negative histopathology test. Adverse pregnancy outcomes investigated included low birth weight (birth weight below 2.5 kg), prematurity (live birth below 37 weeks), and small-for-gestational-age (SGA) (live born with a birth weight below 10th percentile for gestational age and sex). Weighted baseline category logit, log-binomial, and log-Poisson models were used to assess factors associated with placental malaria, and its association with adverse pregnancy outcomes.ResultsAmong 1115 women who had histopathology and DNA PCR performed, 93 (8%) had HP placental infection, and 136 (12%) had the sub-microscopic placental infection. The risk of sub-microscopic placental malaria was greater in women who did not use mosquito prevention methods such as bed nets, fumigation, or mosquito coils (odds ratio (OR) = 1.75; 95% confidence interval (CI): 1.05–2.92; P = 0.03) and in women who were anemic (OR = 1.59; 95% CI: 1.20–2.11; P = 0.001). Women who were underweight had reduced odds of sub-microscopic placental malaria infection (OR = 0.33; 95% CI: 0.17–0.62; P = 0.001). Women who were overweight/obese had 1.48 times higher the odds of HP placental malaria compared to normal weight (OR = 1.48; 95% CI: 1.03–2.11; P = 0.03). HP placental malaria infection was associated with an increased risk of SGA births (RR = 1.30, 95% CI: 0.98–1.72, P = 0.07). In contrast, the sub-microscopic infection was associated with a reduced risk of SGA births (RR = 0.61, 95% CI: 0.43–0.88, P = 0.01). Placental malaria was not associated with low birth weight or prematurity.ConclusionMalaria prevention methods and maternal nutrition status during early pregnancy were important predictors of sub-microscopic placental malaria. More research is needed to understand sub-microscopic placental malaria and the possible mechanisms mediating the association between placental malaria and SGA.

Age-dependent carriage of alleles and haplotypes of Plasmodium falciparum sera5, eba-175, and csp in a region of intense malaria transmission in Uganda

BackgroundThe development of malaria vaccines is constrained by genetic polymorphisms exhibited by Plasmodium falciparum antigens. The project the age-dependent distribution of alleles or haplotypes of three P. falciparum malaria vaccine candidates, Circumsporozoite Protein (csp), Erythrocyte Binding Antigen 175 (eba-175) and Serine Repeat Antigen 5 (sera5) in a region of intense malaria transmission in Uganda.MethodsA cross-sectional study was carried out between August and November 2009 in which 250 study participants were selected from a population of 600. Finger prick blood samples were collected after informed consent from participants below 5 years, 5–10 years, and above 10 years of age. Blood was used for microscopy, RDT and dried blood spots. Plasmodium falciparum DNA was extracted by chelex method. Alleles of sera5 and eba-175 were determined by polymerase chain reaction (PCR) amplification followed by resolution of products by agarose gel electrophoresis. Allele calling was done using gel photographs from ethiduim bromide stained gels. Haplotypes of csp were identified by sequencing 63 PCR products using the P. falciparum 7G8 laboratory strain sequence as a reference. The data were analysed using SPSS 16, EQX for windows and Chi-square test was used to calculate associations (P-values), Excel was used to generate graphs. The BioEdit and NCBI blast software programs were used to analyse the sequences from which csp haplotypes map was constructed.ResultsEba-175 FCR3 (48/178) and CAMP (16/178) alleles were observed, the FCR3 (24/67) allele being predominant among children aged below 5 years old while the CAMP (12/67) allele was predominant among older participants. Sera5 alleles ORI (6/204) and ORII (103/204) were observed in the population, ORII was more prevalent and was significantly associated with age (P values < 0.0001), parasite density (P-value < 0.0001) and clinical outcomes (P value = 0.018). There was marked csp diversity in the Th2/Th3 region. Out of 63 sequences, 16 conformed to the reference strain and one (1/16) was similar to a West African haplotype and the majority (14/16) of the haplotypes were unique to this study region. There was an age-dependent distribution of csp haplotypes with more haplotypes being harbored by children < 5-year of age, (10/16) compared to adults (2/16). Interestingly, the csp haplotype corresponding to 3D7 whose prototypical sequence is identical to the sequence of the leading malaria vaccine candidate RTS, S was not observed.ConclusionThis data suggest that the eba-175 FCR3 allele, sera5 ORII allele, and csp haplotypes are targets of host immunity and under immune selection pressure in Apac District. These molecules could provide alternative malaria vaccine candidates as sub-unit vaccines.

Method for Malaria Diagnosis Based on Extractions of Samples Using Non-Invasive Techniques: An Opportunity for the Nursing Clinical Practice.

Malaria has been for millennia one of the best known and most destructive diseases affecting humans. Its high impact has aroused great interest for the development of new effective and reliable diagnostic techniques. Recently it has been recently published that hairs from mammal hosts are able to capture, hold and finally remove foreign DNA sequences of Leishmania parasites. The aim of this study was to check if Plasmodium falciparum (P. falciparum) DNA remains stable in blood samples deposited in Whatman paper after suffering different transport and storage conditions, and to compare the sensitivity of these results with those offered by thick a smear and Rapid Diagnostic Test, and besides to examine whether P. falciparum DNA would be detected and quantified by Real time quantitative PCR (qPCR) from hairs of people with different types of malaria. P. falciparum Histidine Repeat Protein II (pHRP-II) antigen detection and P. falciparum DNA were detected in 18 of 19 dry blood samples adhered to Whatman paper (94.74%), besides, Plasmodium DNA was also detected in seven out of 19 hair samples analyzed (36.84%), remaining stable until analysis for several months under the exposure to different environmental conditions. Although the sensitivity of PCR for the diagnosis of malaria in hair samples is not as high as blood analysis, the study of Plasmodium DNA presence in blood and hair could constitute a complementary tool with numerous advantages in sample collection, transport and storage. We suggest that the method could be also applied to medical, forensic and paleo-parasitological diagnosis, not only for malaria but also for searching many other pathogens in hair samples.

Polymorphisms of pfcrt, pfmdr1, and K13-propeller genes in imported falciparum malaria isolates from Africa in Guizhou province, China

BackgroundImported falciparum malaria from Africa has become a key public health challenge in Guizhou Province since 2012. Understanding the polymorphisms of molecular markers of drug resistance can guide selection of antimalarial drugs for the treatment of malaria. This study was aimed to analyze the polymorphisms of pfcrt, pfmdr1, and K13-propeller among imported falciparum malaria cases in Guizhou Province, China.MethodFifty-five imported falciparum malaria cases in Guizhou Province during 2012–2016 were included in this study. Their demographic information and filter paper blood samples were collected. Genomic DNA of Plasmodium falciparum was extracted from the blood samples, and polymorphisms of pfcrt, pfmdr1, and K13-propeller were analyzed with nested PCR amplification followed by sequencing. Data were analyzed with the SPSS17.0 software.ResultsThe prevalence of pfcrt K76T, pfmdr1 N86Y, and pfmdr1 Y184F mutation was 56.6, 22.2, and 72.2%, respectively, in imported falciparum malaria cases in Guizhou Province. We detected two mutant haplotypes of pfcrt, IET and MNT, with IET being more commonly found (54.7%), and five mutant haplotypes of pfmdr1, of which NFD was the most frequent (53.7%). There were totally 10 combined haplotypes of pfcrt and pfmdr1, of which the haplotype IETNFD possessed a predominance of 28.8%. In addition, three nonsynonymous mutations (S459T, C469F, and V692L) and two synonymous mutations (R471R and V589V) were detected in K13-propeller, all having prevalence less than 6.0%. In particular, a candidate K13 resistance mutation, C469F, was identified for the first time from Democratic Republic of the Congo with the prevalence of 2.0%.ConclusionsThe high prevalence of IET haplotype of pfcrt and NFD haplotype of pfmdr1 suggests the presence of chloroquine, artemether/lumefantrine, and dihydroartemisinin/piperaquine resistance in these cases. Therefore cautions should be made to artemisinin therapy for P. falciparum in Africa. Continuous monitoring of anti-malarial drug efficacy in imported malaria cases is helpful for optimizing antimalarial drug therapy in Guizhou Province, China.