Novel genetic polymorphisms associated with severe malaria and under selective pressure in North-eastern Tanzania.

Significant selection pressure has been exerted on the genomes of human populations exposed to Plasmodium falciparum infection, resulting in the acquisition of mechanisms of resistance against severe malarial disease. Many host genetic factors, including sickle cell trait, have been associated with reduced risk of developing severe malaria, but do not account for all of the observed phenotypic variation. Identification of novel inherited risk factors relies upon high-resolution genome-wide association studies (GWAS). We present findings of a GWAS of severe malaria performed in a Tanzanian population (n = 914, 15.2 million SNPs). Beyond the expected association with the sickle cell HbS variant, we identify protective associations within two interleukin receptors (IL-23R and IL-12RBR2) and the kelch-like protein KLHL3 (all P<10-6), as well as near significant effects for Major Histocompatibility Complex (MHC) haplotypes. Complementary analyses, based on detecting extended haplotype homozygosity, identified SYNJ2BP, GCLC and MHC as potential loci under recent positive selection. Through whole genome sequencing of an independent Tanzanian cohort (parent-child trios n = 247), we confirm the allele frequencies of common polymorphisms underlying associations and selection, as well as the presence of multiple structural variants that could be in linkage with these SNPs. Imputation of structural variants in a region encompassing the glycophorin genes on chromosome 4, led to the characterisation of more than 50 rare variants, and individually no strong evidence of associations with severe malaria in our primary dataset (P>0.3). Our approach demonstrates the potential of a joint genotyping-sequencing strategy to identify as-yet unknown susceptibility loci in an African population with well-characterised malaria phenotypes. The regions encompassing these loci are potential targets for the design of much needed interventions for preventing or treating malarial disease.

USP38, FREM3, SDC1, DDC, and LOC727982 Gene Polymorphisms and Differential Susceptibility to Severe Malaria in Tanzania.

Populations exposed to Plasmodium falciparum infection develop genetic mechanisms of protection against severe malarial disease. Despite decades of genetic epidemiological research, the sickle cell trait (HbAS) sickle cell polymorphism, ABO blood group, and other hemoglobinopathies remain the few major determinants in severe malaria to be replicated across different African populations and study designs. Within a case-control study in a region of high transmission in Tanzania (n = 983), we investigated the role of 40 new loci identified in recent genome-wide studies. In 32 loci passing quality control procedures, we found polymorphisms in USP38, FREM3, SDC1, DDC, and LOC727982 genes to be putatively associated with differential susceptibility to severe malaria. Established candidates explained 7.4% of variation in severe malaria risk (HbAS polymorphism, 6.3%; α-thalassemia, 0.3%; ABO group, 0.3%; and glucose-6-phosphate dehydrogenase deficiency, 0.5%) and the new polymorphisms, another 4.3%. The regions encompassing the loci identified are promising targets for the design of future treatment and control interventions.

Effect of context on respiratory rate measurement in identifying non-severe pneumonia in African children.

OBJECTIVE: Cough or difficult breathing and an increased respiratory rate for their age are the commonest indications for outpatient antibiotic treatment in African children. We aimed to determine whether respiratory rate was likely to be transiently raised by a number of contextual factors in a busy clinic leading to inaccurate diagnosis. METHODS: Respiratory rates were recorded in children aged 2-59 months presenting with cough or difficulty breathing to one of the two busy outpatient clinics and then repeated at 10-min intervals over 1 h in a quiet setting. RESULTS: One hundred and sixty-seven children were enrolled with a mean age of 7.1 (SD ± 2.9) months in infants and 27.6 (SD ± 12.8) months in children aged 12-59 months. The mean respiratory rate declined from 42.3 and 33.6 breaths per minute (bpm) in the clinic to 39.1 and 32.6 bpm after 10 min in a quiet room and to 39.2 and 30.7 bpm (P < 0.001) after 60 min in younger and older children, respectively. This resulted in 11/13 (85%) infants and 2/15 (13%) older children being misclassified with non-severe pneumonia. In a random effects linear regression model, the variability in respiratory rate within children (42%) was almost as much as the variability between children (58%). Changing the respiratory rates cut-offs to higher thresholds resulted in a small reduction in the proportion of non-severe pneumonia mis-classifications in infants. CONCLUSION: Noise and other contextual factors may cause a transient increase in respiratory rate and consequently misclassification of non-severe pneumonia. However, this effect is less pronounced in older children than infants. Respiratory rate is a difficult sign to measure as the variation is large between and within children. More studies of the accuracy and utility of respiratory rate as a proxy for non-severe pneumonia diagnosis in a busy clinic are needed.

African glucose-6-phosphate dehydrogenase alleles associated with protection from severe malaria in heterozygous females in Tanzania.

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers.

Candidate human genetic polymorphisms and severe malaria in a Tanzanian population.

Human genetic background strongly influences susceptibility to malaria infection and progression to severe disease and death. Classical genetic studies identified haemoglobinopathies and erythrocyte-associated polymorphisms, as protective against severe disease. High throughput genotyping by mass spectrometry allows multiple single nucleotide polymorphisms (SNPs) to be examined simultaneously. We compared the prevalence of 65 human SNP's, previously associated with altered risk of malaria, between Tanzanian children with and without severe malaria. Five hundred children, aged 1-10 years, with severe malaria were recruited from those admitted to hospital in Muheza, Tanzania and compared with matched controls. Genotyping was performed by Sequenom MassArray, and conventional PCR was used to detect deletions in the alpha-thalassaemia gene. SNPs in two X-linked genes were associated with altered risk of severe malaria in females but not in males: heterozygosity for one or other of two SNPs in the G6PD gene was associated with protection from all forms of severe disease whilst two SNPs in the gene encoding CD40L were associated with respiratory distress. A SNP in the adenyl cyclase 9 (ADCY9) gene was associated with protection from acidosis whilst a polymorphism in the IL-1α gene (IL1A) was associated with an increased risk of acidosis. SNPs in the genes encoding IL-13 and reticulon-3 (RTN3) were associated with increased risk of cerebral malaria. This study confirms previously known genetic associations with protection from severe malaria (HbS, G6PD). It identifies two X-linked genes associated with altered risk of severe malaria in females, identifies mutations in ADCY9, IL1A and CD40L as being associated with altered risk of severe respiratory distress and acidosis, both of which are characterised by high serum lactate levels, and also identifies novel genetic associations with severe malaria (TRIM5) and cerebral malaria(IL-13 and RTN3). Further studies are required to test the generality of these associations and to understand their functional consequences.

WHO guidelines for antimicrobial treatment in children admitted to hospital in an area of intense Plasmodium falciparum transmission: prospective study.

OBJECTIVES: To assess the performance of WHO's "Guidelines for care at the first-referral level in developing countries" in an area of intense malaria transmission and identify bacterial infections in children with and without malaria. DESIGN: Prospective study. SETTING: District hospital in Muheza, northeast Tanzania. PARTICIPANTS: Children aged 2 months to 13 years admitted to hospital for febrile illness. MAIN OUTCOME MEASURES: Sensitivity and specificity of WHO guidelines in diagnosing invasive bacterial disease; susceptibility of isolated organisms to recommended antimicrobials. RESULTS: Over one year, 3639 children were enrolled and 184 (5.1%) died; 2195 (60.3%) were blood slide positive for Plasmodium falciparum, 341 (9.4%) had invasive bacterial disease, and 142 (3.9%) were seropositive for HIV. The prevalence of invasive bacterial disease was lower in slide positive children (100/2195, 4.6%) than in slide negative children (241/1444, 16.7%). Non-typhi Salmonella was the most frequently isolated organism (52/100 (52%) of organisms in slide positive children and 108/241 (45%) in slide negative children). Mortality among children with invasive bacterial disease was significantly higher (58/341, 17%) than in children without invasive bacterial disease (126/3298, 3.8%) (P<0.001), and this was true regardless of the presence of P falciparum parasitaemia. The sensitivity and specificity of WHO criteria in identifying invasive bacterial disease in slide positive children were 60.0% (95% confidence interval 58.0% to 62.1%) and 53.5% (51.4% to 55.6%), compared with 70.5% (68.2% to 72.9%) and 48.1% (45.6% to 50.7%) in slide negative children. In children with WHO criteria for invasive bacterial disease, only 99/211(47%) of isolated organisms were susceptible to the first recommended antimicrobial agent. CONCLUSIONS: In an area exposed to high transmission of malaria, current WHO guidelines failed to identify almost a third of children with invasive bacterial disease, and more than half of the organisms isolated were not susceptible to currently recommended antimicrobials. Improved diagnosis and treatment of invasive bacterial disease are needed to reduce childhood mortality.

A screening instrument to measure the prevalence of neurological disability in resource-poor settings.

BACKGROUND: Very little is known about the prevalence of neurological morbidity in Africa. Much of this is due to the difficulty in performing epidemiological surveys in these settings. A screening instrument to measure neurological disease in resource-poor settings was designed by the World Health Organization in 1981, but several problems with it have subsequently been recognized. METHODS: We created a new screening instrument that addressed problems with the original instrument identified by prior investigators, and that included questions to identify diseases of public health significance and broad neurological syndromes. This new instrument was tested in an outpatient setting in Moshi, Tanzania. We compared the sensitivity and specificity of the new instrument to the original 1981 WHO instrument. RESULTS: We tested the survey on 128 participants. Of these, 63 had neurological diagnoses, 21 had pain-only diagnoses and 44 had no neurological diagnoses. The survey was well received by all the participants. A nonmedical interviewer was trained to administer and interpret a simple neurological examination without difficulty. The median time to administer the instrument was 13 min (interquartile range 10-17 min). The sensitivity of the new instrument improved that of the WHO instrument from 98.4 to 100%, but the difference was not statistically significant (p = 0.44). However, the specificity significantly improved from 29.2 to 61.0% (p = 0.001). CONCLUSIONS: We have developed a screening instrument to measure the prevalence of neurological morbidity in resource-poor settings. It was shown to be highly feasible, highly sensitive, and more specific than the existing WHO instrument.