Regina N. Mugri

Reappraisal of known malaria resistance loci in a large multicenter study

Many human genetic associations with resistance to malaria have been reported, but few have been reliably replicated. We collected data on 11,890 cases of severe malaria due to Plasmodium falciparum and 17,441 controls from 12 locations in Africa, Asia and Oceania. We tested 55 SNPs in 27 loci previously reported to associate with severe malaria. There was evidence of association at P < 1 × 10−4 with the HBB, ABO, ATP2B4, G6PD and CD40LG loci, but previously reported associations at 22 other loci did not replicate in the multicenter analysis. The large sample size made it possible to identify authentic genetic effects that are heterogeneous across populations or phenotypes, with a striking example being the main African form of G6PD deficiency, which reduced the risk of cerebral malaria but increased the risk of severe malarial anemia. The finding that G6PD deficiency has opposing effects on different fatal complications of P. falciparum infection indicates that the evolutionary origins of this common human genetic disorder are more complex than previously supposed.

Severe and uncomplicated falciparum malaria in children from three regions and three ethnic groups in Cameroon: prospective study

BackgroundTo identify the factors that account for differences in clinical outcomes of malaria as well as its relationship with ethnicity, transmission intensity and parasite density.MethodsA prospective study was conducted in nine health facilities in the Centre, Littoral and South West regions of Cameroon, and in three ethnic groups; the Bantu, Semi-Bantu and Foulbe. Children aged one month to 13 years, with diagnosis suggestive of malaria, were recruited and characterized using the WHO definition for severe and uncomplicated malaria. Malaria parasitaemia was determined by light microscopy, haematological analysis using an automated haematology analyser and glucose level by colorimetric technique.ResultsOf the febrile children screened, 971 of the febrile children screened fulfilled the inclusion criteria for specific malaria clinical phenotypes. Forty-nine (9.2%) children had cerebral malaria, a feature that was similar across age groups, ethnicity and gender but lower (P < 0.004) in proportion in the Centre (3.1%, 5/163) compared to the Littoral (11.3%, 32/284) and South West (13.6%, 12/88) regions. Severe anaemia was the most frequent severe disease manifestation, 28.0% (248/885), which was similar in proportion across the three ethnic groups but was more prevalent in females, less than 60 months old, and the Centre region. About 20% (53/267) of the participants presented with respiratory distress, a clinical phenotype independent of age, gender and ethnicity, but highest (P < 0.001) in the Centre (55%, 11/20) compared to the Littoral (27.3%, 3/11) and South West (16.5%, 39/236) regions. Uncomplicated malaria constituted 27.7% (255/920) of hospital admissions and was similar in proportion with gender and across the three ethnic groups but more prevalent in older children (≥ 60 months) as well as in the South West region. The density of malaria parasitaemia was generally similar across clinical groups, gender and ethnicity. However, younger children and residents of the Centre region carried significantly higher parasite loads, with the burden heavier in the Semi-Bantu compared to their Bantu (P = 0.009) and Foulbe (P = 0.026) counterparts in the Centre region. The overall study case fatality was 4.8 (47/755), with cerebral malaria being the only significant risk factor associated with death. Severe anaemia, though a common and major clinical presentation, was not significantly associated with risk of death.ConclusionAbout half of the acutely febrile children presented with severe malaria, the majority being cases of severe malaria anaemia, followed by respiratory distress and cerebral malaria. The latter two were less prevalent in the Centre region compared to the other regions. Cerebral malaria and hyperpyrexia were the only significant risk factors associated with death.

Antenatal care visit attendance, intermittent preventive treatment during pregnancy (IPTp) and malaria parasitaemia at delivery

BackgroundThe determinants and barriers for delivery and uptake of IPTp vary with different regions in sub-Saharan Africa. This study evaluated the determinants of ANC clinic attendance and IPTp-SP uptake among parturient women from Mount Cameroon Area and hypothesized that time of first ANC clinic attendance could influence uptake of IPTp-SP/dosage and consequently malaria parasite infection status at delivery.MethodsTwo cross sectional surveys were carried out at the Government Medical Centre in the Mutengene Health Area, Mt Cameroon Area from March to October 2007 and June 2008 to April 2009. Consented parturient women were consecutively enrolled in both surveys. In 2007, socio-demographic data, ANC clinic attendance, gestational age, fever history and reported use/dosage of IPTp-SP were documented using a structured questionnaire. In the second survey only IPT-SP usage/dosage was recorded. Malaria parasitaemia at delivery was determined by blood smear microscopy and placental histology.Results and discussionIn 2007, among the 287 women interviewed, 2.2%, 59.7%, and 38.1% enrolled in the first, second and third trimester respectively. About 90% of women received at least one dose SP but only 53% received the two doses in 2007 and by 2009 IPTp-two doses coverage increased to 64%. Early clinic attendance was associated (P = 0.016) with fever history while being unmarried (OR = 2.2; 95% CI: 1.3-3.8) was significantly associated with fewer clinic visits (<4visits). Women who received one SP dose (OR = 3.7; 95% CI: 2.0-6.8) were more likely not to have attended ≥ 4visits. A higher proportion (P < 0.001) of women with first visit during the third trimester received only one dose, meanwhile, those who had an early first ANC attendance were more likely (OR = 0.4; 95% CI = 0.2 - 0.7) to receive two or more doses. Microscopic parasitaemia at delivery was frequent (P = 0.007) among women who enrolled in the third trimester and had received only one SP dose than in those with two doses.ConclusionIn the study area, late first ANC clinic enrolment and fewer clinic visits may prevent the uptake of two SP doses and education on early and regular ANC clinic visits can increase IPTp coverage.

Association of Cytokine and Toll-Like Receptor Gene Polymorphisms with Severe Malaria in Three Regions of Cameroon

P. falciparum malaria is one of the most widespread and deadliest infectious diseases in children under five years in endemic areas. The disease has been a strong force for evolutionary selection in the human genome, and uncovering the critical human genetic factors that confer resistance to the disease would provide clues to the molecular basis of protective immunity that would be invaluable for vaccine development. We investigated the effect of single nucleotide polymorphisms (SNPs) on malaria pathology in a case- control study of 1862 individuals from two major ethnic groups in three regions with intense perennial P. falciparum transmission in Cameroon. Twenty nine polymorphisms in cytokine and toll-like receptor (TLR) genes as well as the sickle cell trait (HbS) were assayed on the Sequenom iPLEX platform. Our results confirm the known protective effect of HbS against severe malaria and also reveal a protective effect of SNPs in interleukin-10 (IL10) cerebral malaria and hyperpyrexia. Furthermore, IL17RE rs708567 GA and hHbS rs334 AT individuals were associated with protection from uncomplicated malaria and anaemia respectively in this study. Meanwhile, individuals with the hHbS rs334 TT, IL10 rs3024500 AA, and IL17RD rs6780995 GA genotypes were more susceptible to severe malarial anaemia, cerebral malaria, and hyperpyrexia respectively. Taken together, our results suggest that polymorphisms in some immune response genes may have important implications for the susceptibility to severe malaria in Cameroonians. Moreover using uncomplicated malaria may allow us to identify novel pathways in the early development of the disease.

Characterisation of the opposing effects of G6PD deficiency on cerebral malaria and severe malarial anaemia

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is believed to confer protection against Plasmodium falciparum malaria, but the precise nature of the protective effect has proved difficult to define as G6PD deficiency has multiple allelic variants with different effects in males and females, and it has heterogeneous effects on the clinical outcome of P. falciparum infection. Here we report an analysis of multiple allelic forms of G6PD deficiency in a large multi-centre case-control study of severe malaria, using the WHO classification of G6PD mutations to estimate each individual’s level of enzyme activity from their genotype. Aggregated across all genotypes, we find that increasing levels of G6PD deficiency are associated with decreasing risk of cerebral malaria, but with increased risk of severe malarial anaemia. Models of balancing selection based on these findings indicate that an evolutionary trade-off between different clinical outcomes of P. falciparum infection could have been a major cause of the high levels of G6PD polymorphism seen in human populations. DOI: http://dx.doi.org/10.7554/eLife.15085.001

The effect of Insecticide Treated Nets (ITNs) on Plasmodium falciparum infection in rural and semi-urban communities in the south west region of Cameroon.

Insecticide Treated Nets (ITNs) have been shown to reduce morbidity and mortality, but coverage and proper utilization continues to be moderate in many parts of sub-Saharan Africa. The gains made through a nationwide free distribution were explored as well as the effect on malaria prevalence in semi-urban and rural communities in south western Cameroon. A cross sectional survey was conducted between August and December 2013. Information on net possession, status and use were collected using a structured questionnaire while malaria parasitaemia was determined on Giemsa-stained blood smears by light microscopy. ITN ownership increased from 41.9% to 68.1% following the free distribution campaign, with 58.3% (466/799) reportedly sleeping under the net. ITN ownership was lower in rural settings (adjusted OR = 1.93, 95%CI = 1.36–2.74, p<0.001) and at lower altitude (adjusted OR = 1.79, 95%CI = 1.22–2.62, p = 0.003) compared to semi-urban settings and intermediate altitude respectively. Conversely, ITN usage was higher in semi-urban settings (p = 0.002) and at intermediate altitude (p = 0.002) compared with rural localities and low altitude. Malaria parasitaemia prevalence was higher in rural (adjusted OR = 1.63, 95%CI = 1.07–2.49) compared to semi-urban settings and in those below 15 years compared to those 15 years and above. Overall, participants who did not sleep under ITN were more susceptible to malaria parasitaemia (adjusted OR = 1.70, 95%CI = 1.14–2.54, p = 0.009). Despite the free distribution campaign, ITN ownership and usage, though improved, is still low. As children who reside in rural settings have greater disease burden (parasitemia) than children in semi-urban settings, the potential gains on both reducing inequities in ITN possession as well as disease burden might be substantial if equitable distribution strategies are adopted.

Determinants of Infant Susceptibility to Malaria During the First Year of Life in South Western Cameroon

Background. Falciparum malaria is an important pediatric infectious disease that frequently affects pregnant women and alters infant morbidity. However, the impact of some prenatal and perinatal risk factors such as season and intermittent preventive treatment during pregnancy (IPTp) on neonatal susceptibility has not been fully elucidated. Methods. A cohort of 415 infants born to women who were positive and negative for malaria was monitored in a longitudinal study in Southwestern Cameroon. The clinical and malaria statuses were assessed throughout, whereas paired maternal-cord and 1-year-old antimalarial antibodies were assayed by enzyme-linked immunosorbent assay. Infant susceptibility to malaria was ascertained after accounting for IPTp and season in the statistical analysis. Results. Malaria prevalence was higher in women (P = .039) who delivered during the rainy season and their infants (P = .030) compared with their dry season counterparts. Infants born to women who were positive for malaria (6.40 ± 2.83 months) were older (P = .028) than their counterparts who were negative for malaria (5.52 ± 2.85 months) when they experienced their first malaria episode. Infants born in September–November (adjusted odds ratio [OR] = 0.31, 95% confidence interval [CI] = 0.13–0.72) and to mothers on 1 or no IPTp-sulfadoxine/pyrimethamine (SP) dose (adjusted OR = 0.51, 95% CI = 0.28–0.91) were protected, whereas those born in the rainy season (adjusted OR = 2.82, 95% CI = 1.21–6.55) were susceptible to malaria. Conclusions. Intermittent preventive treatment during pregnancy and month of birth have important implications for infant susceptibility to malaria, with 2 or more IPTp-SP dosage possibly reducing immunoglobulin M production.

Association of candidate gene polymorphisms and TGF-beta/IL-10 levels with malaria in three regions of Cameroon: a case-control study

BackgroundPlasmodium falciparum malaria is one of the most widespread and deadliest infectious diseases in children under five years in endemic areas. The disease has been a strong force for evolutionary selection in the human genome, and uncovering the critical host genetic factors that confer resistance to the disease would provide clues to the molecular basis of protective immunity and improve vaccine development initiatives.MethodsThe effect of single nucleotide polymorphisms (SNPs) and plasma transforming growth factor beta (TGF-β) and interleukin 10 (IL-10) levels on malaria pathology was investigated in a case–control study of 1862 individuals from two major ethnic groups in three regions with intense perennial P. falciparum transmission in Cameroon. Thirty-four malaria candidate polymorphisms, including the sickle cell trait (HbS), were assayed on the Sequenom iPLEX platform while plasma TGF-β and IL-10 levels were measured by sandwich ELISA.ResultsThe study confirms the known protective effect of HbS against severe malaria and also reveals a protective effect of SNPs in the nitrogen oxide synthase 2 (NOS2) gene against malaria infection, anaemia and uncomplicated malaria. Furthermore, ADCY9 rs10775349 (additive G) and ABO rs8176746 AC individuals were associated with protection from hyperpyrexia and hyperparasitaemia, respectively. Meanwhile, individuals with the EMR1 rs373533 GT, EMR1 rs461645 CT and RTN3 rs542998 (additive C) genotypes were more susceptible to hyperpyrexia while both females and males with the rs1050828 and rs1050829 SNPs of G6PD, respectively, were more vulnerable to anaemia. Plasma TGF-β levels were strongly correlated with heterozygosity for the ADCY9 rs2230739 and HBB rs334 SNPs while individuals with the ABO rs8176746 AC genotype had lower IL-10 levels.ConclusionTaken together, this study suggests that some rare polymorphisms in candidate genes may have important implications for the susceptibility of Cameroonians to severe malaria. Moreover using the uncomplicated malaria phenotype may permit the identification of novel pathways in the early development of the disease.

Molecular markers for artemisinin and partner drug resistance in natural Plasmodium falciparum populations following increased insecticide treated net coverage along the slope of mount Cameroon: cross-sectional study

BackgroundDrug resistance is one of the greatest challenges of malaria control programmes, with the monitoring of parasite resistance to artemisinins or to Artemisinin Combination Therapy (ACT) partner drugs critical to elimination efforts. Markers of resistance to a wide panel of antimalarials were assessed in natural parasite populations from southwestern Cameroon.MethodsIndividuals with asymptomatic parasitaemia or uncomplicated malaria were enrolled through cross-sectional surveys from May 2013 to March 2014 along the slope of mount Cameroon. Plasmodium falciparum malaria parasitaemic blood, screened by light microscopy, was depleted of leucocytes using CF11 cellulose columns and the parasite genotype ascertained by sequencing on the Illumina HiSeq platform.ResultsA total of 259 participants were enrolled in this study from three different altitudes. While some alleles associated with drug resistance in pfdhfr, pfmdr1 and pfcrt were highly prevalent, less than 3% of all samples carried mutations in the pfkelch13 gene, none of which were amongst those associated with slow artemisinin parasite clearance rates in Southeast Asia. The most prevalent haplotypes were triple mutants PfdhfrI51R59N108I164(99%), pfcrt- C72V73I74E75T76 (47.3%), and single mutants PfdhpsS436G437K540A581A613(69%) and Pfmdr1 N86F184D1246 (53.2%).ConclusionsThe predominance of the Pf pfcrt CVIET andPfdhfrIRN triple mutant parasites and absence of pfkelch13 resistance alleles suggest that the amodiaquine and pyrimethamine components of AS-AQ and SP may no longer be effective in their role while chloroquine resistance still persists in southwestern Cameroon.

THE EFFECT OF HELMINTH CO-INFECTION ON MALARIA-SPECIFIC IMMUNOGLOBULIN G RESPONSES

Background Malaria and helminthiases overlap extensively in their epidemiological distributions, and co-infections are common. Helminth infection has a profound effect on the immune system such as the induction of immuno-regulatory mechanisms such as potent regulatory T cell responses known to suppress cellular effector mechanisms. Methods The prevalence of malaria parasitaemia, intestinal helminths, co-infection and anaemia was determined in a cross-sectional study (March 2011) of 372 children aged 6 months to 10 years resident in Mutengene in south-western Cameroon. Plasma total IgG and IgG1–4 subclass antibody levels to P. falciparum apical membrane antigen 1 (AMA1), the N-terminal non-repeat region (GLURP R0) and the C-terminal repeat region of glutamate rich protein (GLURP R2) and merozoite surface protein 3 (MSP3) were measured by standardised ELISA. Results Prevalence was as follows: malaria parasitaemia (mp) 18%, pyrexia 25.4%, helminths 19.7%, and anaemia 71.5%. Amongst those who were mp-positive, 25.4% were symptomatic (4.5% overall). Almost all helminth infections were the soil-transmitted helminths Ascaris, Trichuris and hookworm (96.4%) with a few cases of Hymenolepis and Enterobius. Haemoglobin concentration (g/dl) correlated positively with age and negatively with mp density (p≤0.001). The mean haemoglobin (g/dl) level of participants co-infected with both parasites (3.4%) was higher compared to participants infected with either Plasmodium (15.8%) or helminths (16.1%) alone (p< 0.01). IgG and IgG1–4 subclass antibody levels to all recombinant antigens correlated positively with age (p< 0.01). Total IgG, IgG1, 2 & 3 levels to all the antigens tested were significantly (except MSP3 IgG2, p=0.08) higher in participants infected with Plasmodium alone, compared to the co-infection, helminths only and no infection groups. Decreased levels of AMA1 IgG associated significantly with co-infection (OR=0.27, 95% CI:0.11–0.68). Increased MSP3 IgG and IgG1–4 levels were significantly associated with children infected with Plasmodium alone compared to children co-infected with both parasites. Conclusions Infection with intestinal helminths stifles protective anti-plasmodial antibody responses.