Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Prevalence of Infection and Malaria Parasite Density among Under Five Children: A Case Study of Dunukofia Rural Community in Anambra State, Nigeria

Aim: Malaria still remains an overwhelming cause of morbidity and mortality among children under five years of age, especially in sub-Saharan Africa. The study was carried out to evaluate malaria prevalence amongst children less than five years old. Study Design: A cross sectional study was carried out. The study adopted a retrospective descriptive survey using the hospital records and diagnostic cross sectional survey by examination of blood samples across three variables: gender, age group and mosquito net usage. Duration: The study was done in 2021 from the month of March to April in the rural community. Methodology: Parasitological diagnosis was with Plasmodium falciparum histidine-rich protein 2-based malaria Rapid Diagnostic Test (RDT) and microscopy of giemsa-stained blood smears. Demographic information was collected using questionnaire. Results: Three hundred (300) children aged less than five years malaria infection status was investigate, 174 (58.00% ) of them were females while 126 (42.00%. ) were males. Twenty one percent (21.00%) of the respondents are <1 year, 23.33% (70) of them are between the ages of 2 to 3 years, while 55.67% (167) were 4 years and above. Current malaria prevalence was higher with microscopy (67.33%) than that of RDT (23.33%). Also, previous RDT results showed that there was a higher prevalence (73.56%) of malaria parasites in females than males (58.73%). The microscopy results showed that males had a higher prevalence (38.10%) of malaria parasites than females (12.64%). Overall gender result also revealed that males had a higher prevalence (96.83%) of malaria parasites than females (86.21%). There was a significant difference in the prevalence result with gender (P<0.05). Females had higher parasite density (28.05±15.390) than males (23.22±19.171), there was no significant difference (P>0.05). It further revealed that children from 4 years and above had higher intensity (29.68±17.357) while those of 1 year and below had the least (14.89±16.069). However, there was no significant difference in the malaria parasite among the age groups of patients (P>0.05). Conclusion: Prevalence of malaria parasitaemia was still high in Dunukaofia, Anamba State, Nigeria despite various control measures and interventions put in place by WHO.

Assessment of Vitamin D Status in Patients Suffering from Uncomplicated Malaria in a Health Center in the District of Abidjan (Côte d’Ivoire)

Aims: The pathophysiology of Plasmodium falciparum infection is most often associated with anemia and immune deficiency. Given the important role of vitamin D in the synthesis of hemoglobin and in the stimulation of the immune system, it would be essential to assess the vitamin D status of patients with malaria in order to improve the quality of treatment management. Methodology: A thick drop and a blood smear were used to determine parasite density and parasite species respectively. The complete blood count was performed using an automated analyzer labelled Sysmex XN 1000i. Biochemical parameters such as calcium and phosphorus were determined using the Cobas C311 Hitachi. The Vidas was used to determine the concentrations of 25 (OH) -vitamin D. Results: The results showed a decrease in 25 (OH) -vitamin D concentrations in relation to the parasite density and anemia observed in patients with uncomplicated malaria. Conclusion: Vitamin D status in patients with uncomplicated malaria could represent an essential biomarker in the monitoring of antimalarial treatment.

Dynamic changes in genetic diversity, drug resistance mutations, and treatment outcomes of falciparum malaria from the low-transmission to the pre-elimination phase on the islands of São Tomé and Príncipe

Background With effective vector control and case management, substantial progress has been made towards eliminating malaria on the islands of São Tomé and Príncipe (STP). This study assessed the dynamic changes in the genetic diversity of Plasmodium falciparum, the anti-malarial drug resistance mutations, and malaria treatment outcomes between 2010 and 2016 to provide insights for the prevention of malaria rebounding. Methods Polymorphic regions of merozoite surface proteins 1 and 2 (msp1 and msp2) were sequenced in 118 dried blood spots (DBSs) collected from malaria patients who had visited the Central Hospital in 2010–2016. Mutations in the multi-drug resistance I (pfmdr1), chloroquine resistance transporter (pfcrt), and kelch 13 (pfk13) genes were analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) and sequencing in 111 DBSs. A total of 7482 cases that completed a 28-day follow-up were evaluated for treatment outcomes based on the microscopic results. Regression models were used to characterize factors associated with levels of parasite density and treatment failures. Results Parasite strains in STP showed significant changes during and after the peak incidence in 2012. The prevalent allelic type in msp1 changed from K1 to MAD20, and that in msp2 changed from 3D7/IC to FC27. The dominant alleles of drug-resistance markers were pfmdr1 86Y, 184F, D1246, and pfcrt 76 T (Y-F-D-T, 51.4%). The average parasite density in malaria cases declined threefold from low-transmission (2010–2013) to pre-elimination period (2014–2016). Logistic regression models showed that patients with younger age (OR for age = 0.97–0.98, p < 0.001), higher initial parasite density (log10-transformed, OR = 1.44, p < 0.001), and receiving quinine treatment (compared to artemisinin-based combination therapy, OR = 1.91–1.96, p < 0.001) were more likely to experience treatment failures during follow-up. Conclusions Plasmodium falciparum in STP had experienced changes in prevalent strains, and increased mutation frequencies in drug-resistance genes from the low-transmission to the pre-elimination settings. Notably, patients with younger age and receiving quinine treatment were more likely to show parasitological treatment failure during follow-up. Therapeutic efficacy should be carefully monitored to inform future treatment policy in STP.

Prevalence of Plasmodium Falciparum among Medical Students with different ABO Groups and Electrophoretic Patterns in a Tertiary Institution in Nnewi, Nigeria

There is evidence that Plasmodium falciparum (Pf) malaria is influenced by ABO blood type but the extent of association is not fully established. Some investigators opinioned that haemoglobin electrophoretic patterns are a factor in susceptibility to Pf infection but there is no consensus on possible association between it and ABO blood group and Hb genotypes. This study was designed to determine the prevalence of Pf among different ABO blood groups and Hb electrophoretic patterns of medical students of a tertiary institution in Nnewi, Nigeria. A total of 80 subjects (41 males and 39 females) aged 18-30 years who reported to the Medical Centre of the institution on account of febrile illness were recruited for the study. Information on age, previous malaria episodes and recent use of prophylaxis were sought. Three milliliters (3ml) of blood were collected into EDTA container for ABO grouping, Hb electrophoresis and blood films for P. falciparum detection and quantification by microscopy. Pf prevalence among the subjects was 47.5% (38/80). Thirty-one (38.75%) of the subjects were of blood group O, 27 (33.75%) group A, 19 (23.75%) blood group B and 3(3.75%) blood group AB. Fifty-two (65%) of the subjects were Hb AA and 28 (35%) AS. No significance difference was seen between malaria episodes and ABO blood groups; Hb electrophoretic patterns; gender and parasite density (p>0.05) respectively. A negative correlation was observed between parasite density and age (r= -0.180, p = 0.109). Pf infection, frequency of infection and parasite load is not influenced by blood group and Hb electrophoretic patterns in our study population.

Myeloperoxidase and Other Markers of Neutrophil Activation Associate With Malaria and Malaria/HIV Coinfection in the Human Placenta

IntroductionPlacental malaria (PM) is characterized by accumulation of inflammatory leukocytes in the placenta, leading to poor pregnancy outcomes. Understanding of the underlying mechanisms remains incomplete. Neutrophils respond to malaria parasites by phagocytosis, generation of oxidants, and externalization of Neutrophil Extracellular Traps (NETs). NETs drive inflammation in malaria but evidence of NETosis in PM has not been reported. Neutrophil activity in the placenta has not been directly investigated in the context of PM and PM/HIV-co-infection.MethodsUsing peripheral and placental plasma samples and placental tissue collected from Kenyan women at risk for malaria and HIV infections, we assessed granulocyte levels across all gravidities and markers of neutrophil activation, including NET formation, in primi- and secundigravid women, by ELISA, western blot, immunohistochemistry and immunofluorescence.ResultsReduced peripheral blood granulocyte numbers are observed with PM and PM/HIV co-infection in association with increasing parasite density and placental leukocyte hemozoin accumulation. In contrast, placental granulocyte levels are unchanged across infection groups, resulting in enhanced placental: peripheral count ratios with PM. Within individuals, PM- women have reduced granulocyte counts in placental relative to peripheral blood; in contrast, PM stabilizes these relative counts, with HIV coinfection tending to elevate placental counts relative to the periphery. In placental blood, indicators of neutrophil activation, myeloperoxidase (MPO) and proteinase 3 (PRTN3), are significantly elevated with PM and, more profoundly, with PM/HIV co-infection, in association with placental parasite density and hemozoin-bearing leukocyte accumulation. Another neutrophil marker, matrix metalloproteinase (MMP9), together with MPO and PRTN3, is elevated with self-reported fever. None of these factors, including the neutrophil chemoattractant, CXCL8, differs in relation to infant birth weight or gestational age. CXCL8 and MPO levels in the peripheral blood do not differ with infection status nor associate with birth outcomes. Indicators of NETosis in the placental plasma do not vary with infection, and while structures consistent with NETs are observed in placental tissue, the results do not support an association with PM.ConclusionsGranulocyte levels are differentially regulated in the peripheral and placental blood in the presence and absence of PM. PM, both with and without pre-existing HIV infection, enhances neutrophil activation in the placenta. The impact of local neutrophil activation on placental function and maternal and fetal health remains unclear. Additional investigations exploring how neutrophil activation and NETosis participate in the pathogenesis of malaria in pregnant women are needed.

Fasting Blood Glucose in a Ghanaian Adult is Causally Linked with Malaria Parasite Count: A Mechanistic Case Study Using Convergent Cross Mapping

Background: Adults with diabetes mellitus (DM) in malaria-endemic areas might be more susceptible to Plasmodium infection than healthy individuals. Herein, we aimed at verifying the hypothesis that increased fasting blood glucose (FBG) promotes parasite growth as reflected by increased parasite density. Methods: Seven adults without DM were recruited in rural Ghana to determine the relationships between FBG and malaria parasite load. Socio-economic data were recorded in questionnaire-based interviews. Over a period of 6 weeks, we measured FBG and Plasmodium spc. infection in peripheral blood samples photometrically and by polymerase chain reaction (PCR)-assays, respectively. Daily physical activity and weather data were documented via smartphone recording. For the complex natural systems of homeostatic glucose control and Plasmodium spc. lifecycle, empirical dynamic modelling was applied. Results: At baseline, four men and three women (median age, 33 years; interquartile range, 30-48) showed a median FBG of 5.5 (5.1-6.0 mmol/L); one participant had an asymptomatic Plasmodium spc. infection (parasite density: 240 /µL). In this participant, convergent cross mapping (CCM) for 34 consecutive days, showed that FBG was causally affected by parasite density (p <0.02), while the reciprocal relationship was not discernible (p >0.05). Additionally, daily ambient temperature affected parasite density (p<0.01).Conclusion: In this study population living in a malaria-endemic area, we successfully piloted time series analyses for the relationships between FBG and Plasmodium spc. density. Longer observation periods and larger samples are required to confirm these findings and determine the direction of causality.

B-lines by lung ultrasound is associated with pulmonary symptoms and cardiac function in acute malaria: a prospective cohort study

Background Malaria patients are at risk of cardiopulmonary complications, but diagnosis and management are difficult in resource limited environments. B-lines by lung ultrasonography (LUS) can identify pulmonary alterations, however, little is known about the usefulness in malaria. Purpose We aimed to investigate the occurrence of B-lines in acute malaria patients at baseline and at follow-up, and whether they are associated with shortness of breath and impaired left ventricular ejection fraction (LVEF). Methods Adult patients with non-severe acute malaria were prospectively enrolled from June to December 2020 in community healthcare clinics in a remote area. Patients were age- and sex-matched to controls without a prior history of malaria. We examined patients prior to anti-malaria treatment and at follow-up. Malaria treatment was administered according to national guidelines. Patients were excluded if they were pregnant, had concomitant infections or recent chest trauma. Patients underwent LUS (8-zones), echocardiography and peripheral blood smear. Measurements were blinded to clinical variables and outcomes. Results We included a total of 99 patients (median age 40±15 years, 55% men). Patients suffered from Plasmodium vivax (n=75), P. falciparum (n=22), and a mix of the two (n=2) and median parasite density was 1,595 parasites/mL (interquartile range [IQR] 528–6,585/mL). Follow-up was completed in 71 patients and the median follow-up time was 31 days (IQR 27–40 days). Patients with acute malaria had significantly more B-lines at baseline than matched controls (P-value&lt;0.001) and fewer B-lines at follow-up (P-value&lt;0.001) (Figure 1). In acute malaria patients, number of B-lines at baseline correlated significantly with shortness of breath (OR 1.20, [1.04 to 1.39], P-value=0.01) and with LVEF (adjusted for age and sex: +8% [+1% to +15%], P-value=0.016 per 1% decrease in LVEF). There was no correlation between number of B-lines and parasite density (+2% [−5% to +11%], P-value=0.53 per 1000 increase in parasite density). Conclusion B-lines detected by LUS are more frequent in patients with acute malaria than in age- and sex-matched controls and decrease in response to treatment. B-lines also correlate with shortness of breath and lower LVEF at baseline. Because LUS is a quick and accessible examination, it may potentially facilitate risk stratification and therapeutic decisions regarding cardiopulmonary complications in patients with acute malaria. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Danish Heart Association

Estimating Malaria Attributable Fractions With Changing Transmission Intensity: Bayesian Latent Class Vs Logistic Models

Background Asymptomatic carriage of malaria parasites is common in high transmission intensity areas and confounds clinical case definitions for research studies. This is important for investigations that aim to identify immune correlates of protection from clinical malaria. The proportion of fevers attributable to malaria parasites is widely used to define different thresholds of parasite density associated with febrile episodes. We investigated whether varying intensity of malaria transmission had a significant impact on parasite density thresholds. We used the same dataset to explore an alternative statistical approach using the probability of developing fevers as a choice over threshold cut-offs as the former has been reported to increase predictive power. Methods Data from children monitored longitudinally between 2005 and 2017 from Junju and Chonyi in Kilifi, Kenya were analysed. We compare the performance of Bayesian-latent class and logistic power models in estimating malaria attributable fractions and probabilities of having fever given a parasite density with changing malaria transmission intensity. Zero-inflated beta regressions were used to assess the impact of using probabilities to evaluate anti-merozoite antibodies as correlates of protection compared with multilevel binary regression.ResultsMalaria transmission intensity declined from over 49% to 5% between 2006 and 2017 respectively. During this period, malaria attributable fraction varied between 27%-59% using logistic regression compared to 10%-36% using the Bayesian latent class approach. Both models estimated similar patterns of fevers attributable to malaria with changing transmission intensities. The former performed well in estimating the probabilities of having fever, while the latter was efficient in determining the parasite density threshold. However, compared to the logistic power model, the Bayesian algorithm yielded lower estimates for both attributable fractions and probabilities of fever. In modelling the association of merozoite antibodies and clinical malaria, both approaches resulted in comparable estimates, but the utilization of probabilities had a better statistical fit. ConclusionsMalaria attributable fractions varied with an overall decline in the malaria transmission intensity in this setting but did not significantly impact the outcomes of analyses aimed at identifying immune correlates of protection. These data confirm the statistical advantage of using probabilities over binary data.