Samuel B. Anyona

Factors associated with non-adherence to Artemisinin-based combination therapy (ACT) to malaria in a rural population from holoendemic region of western Kenya

BackgroundOver the years, reports implicate improper anti-malarial use as a major contributor of morbidity and mortality amongst millions of residents in malaria endemic areas, Kenya included. However, there are limited reports on improper use of Artemisinin-based Combination Therapy (ACT) which is a first-line drug in the treatment of malaria in Kenya. Knowing this is important for ensured sustainable cure rates and also protection against the emergence of resistant malarial parasites. We therefore investigated ACT adherence level, factors associated with non-adherence and accessibility in households (n = 297) in rural location of Southeast Alego location in Siaya County in western Kenya.MethodsACT Adherence level was assessed with reference to the duration of treatment and number of tablets taken. Using systematic random sampling technique, a questionnaire was administered to a particular household member who had the most recent malaria episode (<2 weeks) and used ACT for cure. Parents/caretakers provided information for children aged <13 years. Key Informant Interviews (KIIs) were also conducted with healthcare providers and private dispensing chemist operators.ResultsAdherence to ACT prescription remained low at 42.1% and 57.9% among individuals above 13 and less than 13 years, respectively. Stratification by demographic and socio-economic characteristics in relation to ACT adherence revealed that age (P = 0.011), education level (P < 0.01), ability to read (P < 0.01) and household (HH) monthly income (P = 0.002) significantly affected the level of ACT adherence. Consistently, logistic regression model demonstrated that low age (OR, 0.571, 95% CI, 0.360-0.905; P = 0.017), higher education level (OR, 0.074; 95% CI 0.017-0.322; P < 0.01), ability to read (OR, 0.285, 95% CI, 0.167-0.486; P < 0.01) and higher income (Ksh. > 9000; OR, 0.340; 95% CI, 0.167-0.694; P = 0.003) were associated with ACT adherence. In addition, about 52.9% of the respondents reported that ACT was not always available at the source and that drug availability (P = 0.020) and distance to drug source (P < 0.01) significantly affected accessibility.ConclusionsThis study demonstrates that more than half of those who get ACT prescription do not take recommended dose and that accessibility is of concern. The findings of this study suggest a potential need to improve accessibility and also initiate programmatic interventions to encourage patient-centred care.

Outpatient Upper Respiratory Tract Viral Infections in Children with Malaria Symptoms in Western Kenya

A cross-sectional study was performed in children 5 through 10 years of age presenting to outpatient clinics in Nyanza Province, Kenya, in which nasal swab and blood specimens were collected during the high malaria transmission season. Patients presenting with malaria-like symptoms within 4 days of fever onset were enrolled in the study. Plasmodium parasitemia was determined by blood smear microscopy. Nasal swabs were screened for a panel of respiratory viruses by polymerase chain reaction. Influenza A, rhinoviruses, and other respiratory viruses were detected in 18%, 26%, and 12% of 197 specimens, respectively. Four of 36 patients with influenza A had a positive malaria blood slide, compared with 20 of 52 patients with rhinovirus. A significant burden of disease caused by influenza A in febrile children during the study period was observed, highlighting the need for further research into the burden of influenza disease in regions where malaria is holoendemic.

Malaria prevalence defined by microscopy, antigen detection, DNA amplification and total nucleic acid amplification in a malaria‐endemic region during the peak malaria transmission season

Objectives  To determine the malaria prevalence by microscopy, antigen detection and nucleic acid detection in a defined subpopulation in a Plasmodium falciparum–endemic region during the peak transmission season.

Pitting of malaria parasites and spherocyte formation

BackgroundA high prevalence of spherocytes was detected in blood smears of children enrolled in a case control study conducted in the malaria holoendemic Lake Victoria basin. It was speculated that the spherocytes reflect intraerythrocytic removal of malarial parasites with a concurrent removal of RBC membrane through a process analogous to pitting of intraerythrocytic inclusion bodies. Pitting and re-circulation of RBCs devoid of malaria parasites could be a host mechanism for parasite clearance while minimizing the anaemia that would occur were the entire parasitized RBC removed. The prior demonstration of RBCs containing ring-infected erythrocyte surface antigen (pf 155 or RESA) but no intracellular parasites, support the idea of pitting.MethodsAn in vitro model was developed to examine the phenomenon of pitting and spherocyte formation in Plasmodium falciparum infected RBCs (iRBC) co-incubated with human macrophages. In vivo application of this model was evaluated using blood specimens from patients attending Kisumu Ditrict Hospital. RBCs were probed with anti-RESA monoclonal antibody and a DNA stain (propidium iodide). Flow cytometry and fluorescent microscopy was used to compare RBCs containing both the antigen and the parasites to those that were only RESA positive.ResultsCo-incubation of iRBC and tumor necrosis factor-alpha activated macrophages led to pitting (14% ± 1.31% macrophages with engulfed trophozoites) as opposed to erythrophagocytosis (5.33% ± 0.95%) (P < 0.01). Following the interaction, 26.9% ± 8.1% of the RBCs were spherocytes as determined by flow cytometric reduction in eosin-5-maleimide binding which detects RBC membrane band 3. The median of patient RBCs with pitted parasites (RESA+, PI-) was more than 3 times (95,275/μL) that of RESA+, PI+ RBCs (28,365/μL) (P < 0.01). RBCs with pitted parasites showed other morphological abnormalities, including spherocyte formation.ConclusionIt is proposed that in malaria holoendemic areas where prevalence of asexual stage parasites approaches 100% in children, RBCs with pitted parasites are re-circulated and pitting may produce spherocytes.

Polymorphic variability in the 3' untranslated region (UTR) of IL12B is associated with susceptibility to severe anaemia in Kenyan children with acute Plasmodium falciparum malaria.

BackgroundPlasmodium falciparum malaria remains a leading cause of morbidity and mortality among African children. Innate immunity provides the first line of defence against P. falciparum infections, particularly in young children that lack naturally-acquired malarial immunity, such as the population examined here. Consistent with the fact that elevated interleukin (IL)-12 is an important component of the innate immune response that provides protective immunity against malaria, we have previously shown that suppression of IL-12 in African children is associated with the development of severe malarial anaemia (SMA). Since the role of IL12B variants in conditioning susceptibility to SMA remains largely unexplored, the association between a single nucleotide polymorphism (1188A→C, rs3212227), SMA (Hb<6.0g/dL), circulating IL-12p40/p70 levels, and longitudinal clinical outcomes in Kenyan children (n = 756) residing in a holoendemic falciparum malaria transmission area were investigated.ResultsMultivariate logistic regression analysis in children with acute malaria (n = 544) demonstrated that carriers of the C allele had increased susceptibility to SMA (CC: OR, 1.674; 95% CI, 1.006-2.673; P = 0.047, and AC: OR, 1.410; 95% CI, 0.953-2.087; P = 0.086) relative to wild type (AA). Although children with SMA had lower IL-12p40/p70 levels than the non-SMA group (P = 0.037), levels did not differ significantly according to genotype. Longitudinal analyses in the entire cohort (n = 756) failed to show any significant relationships between rs3212227 genotypes and either susceptibility to SMA or all-cause mortality throughout the three year follow-up.ConclusionThe rs3212227 is a marker of susceptibility to SMA in children with acute disease, but does not appear to mediate functional changes in IL-12 production or longitudinal outcomes during the acquisition of naturally-acquired malarial immunity.

Functional Promoter Haplotypes of Interleukin-18 Condition Susceptibility to Severe Malarial Anemia and Childhood Mortality

ABSTRACT Severe malarial anemia (SMA) is a leading cause of morbidity and mortality in children residing in regions where Plasmodium falciparum transmission is holoendemic. Although largely unexplored in children with SMA, interleukin-18 (IL-18) is important for regulating innate and acquired immunity in inflammatory and infectious diseases. As such, we selected two functional single-nucleotide polymorphisms (SNPs) in the IL-18 promoter (−137G→C [rs187238] and −607C→A [rs1946518]) whose haplotypes encompass significant genetic variation due to the presence of strong linkage disequilibrium among these variants. The relationship between the genotypes/haplotypes, SMA (hemoglobin [Hb], <5.0 g/dl], and longitudinal clinical outcomes were then investigated in Kenyan children (n = 719). Multivariate logistic regression analyses controlling for age, gender, sickle cell trait, glucose-6-phosphate dehydrogenase (G6PD) deficiency, HIV-1, and bacteremia revealed that carriage of the −607AA genotype was associated with protection against SMA (odds ratio [OR] = 0.440 [95% confidence interval {CI} = 0.21 to 0.90], P = 0.031) in children with acute infection. In contrast, carriers of the −137G/−607C (GC) haplotype had increased susceptibility to SMA (OR = 2.050 [95% CI = 1.04 to 4.05], P = 0.039). Measurement of IL-18 gene expression in peripheral blood leukocytes demonstrated that elevated IL-18 transcripts were associated with reduced hemoglobin concentrations (ρ = −0.293, P = 0.010) and that carriers of the “susceptible” GC haplotype had elevated IL-18 transcripts (P = 0.026). Longitudinal investigation of clinical outcomes over a 3-year follow-up period revealed that carriers of the rare CC haplotype (∼1% frequency) had 5.76 times higher mortality than noncarriers (P = 0.001). Results presented here demonstrate that IL-18 promoter haplotypes that condition elevated IL-18 gene products during acute infection are associated with increased risk of SMA. Furthermore, carriage of the rare CC haplotype significantly increases the risk of childhood mortality.

Interleukin (IL)-13 promoter polymorphisms (-7402 T/G and -4729G/A) condition susceptibility to pediatric severe malarial anemia but not circulating IL-13 levels

In holoendemic Plasmodium falciparum transmission areas such as western Kenya, severe malarial anemia [SMA, hemoglobin (Hb) < 6.0 g/dL, with any density parasitemia] is the most common clinical manifestation of severe malaria resulting in high rates of pediatric morbidity and mortality in these regions. Previous studies associated interleukin (IL)-13 with pathogenesis of different infectious diseases, including P. falciparum malaria. However, the functional roles of polymorphic variants within the IL-13 promoter in conditioning susceptibility to SMA remain largely unexplored. As such, the association between the IL-13 variants -7402 T/G (rs7719175) and -4729G/A (rs3091307) and susceptibility to SMA was determined in children (n = 387) presenting with clinical symptoms of falciparum malaria and resident in a holoendemic transmission region in western Kenya. Our results indicated no difference in the proportions of individual genotypes among children presenting with non-SMA (n = 222) versus SMA (n = 165). Similarly, there was no associations between the individual genotypes (-7402 T/G and -4729G/A) and SMA. Additional analyses, however, revealed that proportions of individuals with -7402 T/-4729A (TA) haplotype was significantly higher in children presenting with SMA than non-SMA group (P = 0.043). A further multivariate logistic regression analyses, controlling for confounding factors, demonstrated that carriage of the TA haplotype was associated with increased susceptibility to SMA (OR; 1.564, 95% CI; 1.023-2.389, P = 0.039). In addition, circulating levels of IL-13 were comparable between the clinical groups as well as across genotypes and haplotypes. Collectively, findings presented here suggest that haplotypes within the IL-13 promoter at -7402 T/G and -4729G/A may modulate SMA pathogenesis, but do not affect circulating IL-13 levels.

Reduced systemic bicyclo‐prostaglandin‐E2 and cyclooxygenase‐2 gene expression are associated with inefficient erythropoiesis and enhanced uptake of monocytic hemozoin in children with severe malarial anemia

In holoendemic Plasmodium falciparum transmission areas, severe malaria primarily occurs in children aged <48 months and manifests as severe malarial anemia [SMA; hemoglobin (Hb) < 6.0 g/dL]. Induction of high levels of prostaglandin‐E2 (PGE2) through inducible cyclooxygenase‐2 (COX‐2) is an important host‐defense mechanism against invading pathogens. We have previously shown that COX‐2‐derived PGE2 levels are reduced in children residing in hyperendemic transmission regions with cerebral malaria and in those with mixed sequelae of anemia and hyperparasitemia. Our in vitro studies further demonstrated that reduced PGE2 was due to downregulation of COX‐2 gene products following phagocytosis of malarial pigment (hemozoin, PfHz). However, as COX‐2‐PGE2 pathways and the impact of naturally acquired PfHz on erythropoietic responses have not been determined in children with SMA, plasma and urinary bicyclo‐PGE2/creatinine and leukocytic COX‐2 transcripts were determined in parasitized children (<36 months) stratified into SMA (n = 36) and non‐SMA (Hb ≥ 6.0 g/dL; n = 38). Children with SMA had significantly reduced plasma (P = 0.001) and urinary (P < 0.001) bicyclo‐PGE2/creatinine and COX‐2 transcripts (P = 0.007). There was a significant positive association between Hb and both plasma (r = 0.363, P = 0.002) and urinary (r = 0.500, P = 0.001)] bicyclo‐PGE2/creatinine. Furthermore, decreased systemic bicyclo‐PGE2/creatinine was associated with inefficient erythropoiesis (i.e., reticulocyte production index; RPI < 2.0, P = 0.026). Additional analyses demonstrated that plasma (P = 0.031) and urinary (P = 0.070) bicyclo‐PGE2/creatinine and COX‐2 transcripts (P = 0.026) progressively declined with increasing concentrations of naturally acquired PfHz by monocytes. Results presented here support a model in which reduced COX‐2‐derived PGE2, driven in part by naturally acquired PfHz by monocytes, promotes decreased erythropoietic responses in children with SMA. Am. J. Hematol., 2012.

Suppressed circulating bicyclo-PGE2 levels and leukocyte COX-2 transcripts in children co-infected with P. falciparum malaria and HIV-1 or bacteremia.

In holoendemic Plasmodium falciparum transmission regions, malarial anemia is a leading cause of childhood morbidity and mortality. Identifying biomarkers of malaria disease severity is important for identifying at-risk groups and for improved understanding of the molecular pathways that influence clinical outcomes. We have previously shown that decreased cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) levels are associated with enhanced clinical severity in cerebral malaria, malarial anemia, and malaria during pregnancy. Since children with malaria often have increased incidence of additional infections, such as bacteremia and HIV-1, we extend our previous findings by investigating COX-2 and PGE2 in children with falciparum malaria and co-infection with either bacteremia or HIV-1. Plasma bicyclo-PGE2/creatinine levels and peripheral blood COX-2 transcripts were significantly reduced in co-infected children relative to those with malaria mono-infection. Furthermore, suppression of circulating bicyclo-PGE2 was significantly associated with reduced hemoglobin levels in both mono- and co-infected children with malaria, suggesting that bicyclo-PGE2 may represent both a marker and mediator of malaria pathogenesis.