ROLE OF MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) AND MIF PROMOTER POLYMORPHISMS IN THE PATHOGENESIS OF SEVERE MALARIAL ANEMIA

Severe malarial anemia (SMA), caused by infections with Plasmodium falciparum, is one of the leading causes of childhood mortality in sub-Saharan Africa. Although the molecular determinants of SMA are largely undefined, dysregulation in host-derived inflammatory mediators influences disease severity. Macrophage migration inhibitory factor (MIF) is an important regulator of innate inflammatory responses that has recently been shown to suppress erythropoiesis and promote pathogenesis of SMA in murine models. The role of MIF in childhood malarial pathogenesis was investigated by examining peripheral blood MIF production in children residing in a hyperendemic area of Gabon, and a holoendemic region of western Kenya. The relationship between MIF concentrations and monocytic acquisition of hemozoin, and the effects of MIF on erythropoiesis in vivo and in vitro were investigated. In addition, the influence of genetic variation at MIF -173 (G/C) and -794 (CATT5-8) on MIF production and susceptibility to SMA and high-density parasitemia (HDP) was examined. Circulating MIF concentrations and peripheral blood mononuclear cells (PBMC) MIF production progressively declined with increasing anemia severity and increasing levels of hemozoin-containing monocytes. However, circulating MIF concentrations were not significantly associated with reticulocyte production in children with acute malaria. Additional experiments in malaria-naïve individuals demonstrated that hemozoin caused both increased and decreased MIF production in cultured PBMC based on genetic differences. In addiiton, a novel in vitro model of erythropoiesis was developed and used to demonstrate that treatment with exogenous MIF or blocking endogenous MIF did not signifcantly impact on the efficiency of erythropoiesis. Genetic analyses revealed that the MIF -173 CC genotype was associated with an increased risk of HDP compared to MIF -173 GG. In addition, individuals with the MIF -794CATT6/-173G haplotype were significantly protected from SMA while those with -794CATT7/8/-173C haplotypes were at an increased risk of developing SMA. Taken together, our findings demonstrate that SMA is associated with decreased MIF production and that individuals with high MIF-producing genetic variants are less susceptible to severe malaria. The public health significance of this study is that investigations presented here increase our understanding of protective inflammatory responses to childhood malaria, which is critical in the formulation of an effective malarial vaccine

A barcode of multilocus nuclear DNA identifies genetic relatedness in pre- and post-Artemether/Lumefantrine treated Plasmodium falciparum in Nigeria.

BACKGROUND: The decline in the efficacy of artemisinin-based combination treatment (ACT) in some endemic regions threatens the progress towards global elimination of malaria. Molecular surveillance of drug resistance in malaria-endemic regions is vital to detect the emergence and spread of mutant strains. METHODS: We observed 89 malaria patients for the efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum infections in Lagos, Nigeria and determined the prevalence of drug resistant strains in the population. Parasite clearance rates were determined by microscopy and the highly sensitive var gene acidic terminal sequence (varATS) polymerase chain reaction for 65 patients with samples on days 0, 1, 3, 7, 14, 21 and 28 after commencement of treatment. The genomic finger print of parasite DNA from pre- and post-treatment samples were determined using 24 nuclear single nucleotide polymorphisms (SNP) barcode for P. falciparum. Drug resistance associated alleles in chloroquine resistance transporter gene (crt-76), multidrug resistance genes (mdr1-86 and mdr1-184), dihydropteroate synthase (dhps-540), dihydrofolate reductase (dhfr-108) and kelch domain (K-13580) were genotyped by high resolution melt analysis of polymerase chain reaction (PCR) fragments. RESULTS: By varATS qPCR, 12 (18.5%) of the participants had detectable parasite DNA in their blood three days after treatment, while eight (12.3%) individuals presented with genotypable day 28 parasitaemia. Complexity of infection (CoI) was 1.30 on day 0 and 1.34 on day 28, the mean expected heterozygosity (HE) values across all barcodes were 0.50 ± 0.05 and 0.56 ± 0.05 on days 0 and 28 respectively. Barcode (π) pairwise comparisons showed high genetic relatedness of day 0 and day 28 parasite isolates in three (37.5%) of the eight individuals who presented with re-appearing infections. Crt-76 mutant allele was present in 38 (58.5%) isolates. The mdr1-86 mutant allele was found in 56 (86.2%) isolates. No mutation in the K-13580 was observed. CONCLUSIONS: Persistence of DNA-detectable parasitaemia in more than 18% of cases after treatment and indications of genetic relatedness between pre- and post-treatment infections warrants further investigation of a larger population for signs of reduced ACT efficacy in Nigeria

Serum biochemical parameters and cytokine profiles associated with natural African trypanosome infections in cattle.

BACKGROUND: Animal African trypanosomiasis (AAT) greatly affects livestock production in sub-Saharan Africa. In Ghana prevalence of AAT is estimated to range between 5 and 50%. Studies have reported serum biochemical aberrations and variability in cytokine profiles in animals during infection. However, information regarding the biochemical parameters and cytokine profiles associated with natural infections are limited. This study was therefore aimed at investigating changes in the levels of serum biochemical parameters and inflammatory cytokines during a natural infection. METHODS: Nested internal transcribed spacer (ITS)-based PCR and sequencing were used to characterise trypanosome infection in cattle at two areas in Ghana (Adidome and Accra) of different endemicities. The cattle were sampled at four to five-week intervals over a period of six months. Levels of serum biochemical parameters, including creatinine, cholesterol, alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bilirubin and total protein and cytokines (interleukin 10, interleukin 4, interleukin 12, interferon gamma and tumor necrosis factor alpha) were measured in serum samples and then compared between infected cattle and uninfected controls. RESULTS: The predominant trypanosome species detected in Accra (non-endemic) and Adidome (endemic) were Trypanosoma theileri and Trypanosoma vivax, respectively. Serum biochemical parameters were similar between infected and uninfected cattle in Accra. Infected cattle at Adidome however, had significantly higher levels of ALP, creatinine, total protein and total bilirubin (P < 0.05) and significantly lower levels of cholesterol (P < 0.05) at specific time points. At basal levels and during infection, significantly higher pro-inflammatory to anti-inflammatory (Th1/Th2) cytokine ratios were observed in cattle at Adidome compared to Accra (P < 0.05), indicating a shift towards Th1 immune response in Adidome. Levels of IL-10 were, however, significantly elevated in infected cattle in Accra (P < 0.05), suggesting high anti-inflammatory cytokine response in Accra. CONCLUSION: These results suggests that cattle in an endemic area repeatedly infected with trypanosomes of different species or different antigenic types demonstrate high pro-inflammatory (Th1) immune response and biochemical alterations whereas cattle in a non-endemic area with predominantly chronic T. theileri infections demonstrate high anti-inflammatory response and no biochemical alterations

Impact of malaria and hepatitis B co-infection on clinical and cytokine profiles among pregnant women.

BackgroundThe overlap of malaria and chronic hepatitis B (CHB) is common in endemic regions, however, it is not known if this co-infection could adversely influence clinical and immunological responses. This study investigated these interactions in pregnant women reporting to antenatal clinics in Ghana.MethodsClinical parameters (hemoglobin, liver function biomarker, peripheral malaria parasitemia, and hepatitis B viremia) and cytokine profiles were assayed and compared across four categories of pregnant women: un-infected, mono-infected with Plasmodium falciparum (Malaria group), mono-infected with chronic hepatitis B virus (CHB group) and co-infected (Malaria+CHB group).ResultsWomen with Malaria+CHB maintained appreciably normal hemoglobin levels (mean±SEM = 10.3±0.3 g/dL). That notwithstanding, Liver function test showed significantly elevated levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin [PConclusionPut together the findings suggests that Malaria+CHB could exacerbate inflammatory cytokine responses and increase susceptibility to liver injury among pregnant women in endemic settings

Prevalence of malaria and hepatitis B among pregnant women in Northern Ghana: Comparing RDTs with PCR.

BackgroundHigh prevalence of malaria and hepatitis B has been reported among pregnant women in Ghana. In endemic areas, the diagnoses of malaria and hepatitis B among pregnant women on antenatal visits are done using histidine-rich protein 2 (HRP2) and hepatitis B surface antigen (HBsAg) rapid diagnostic tests (RDTs), respectively, which are, however, reported to give some false positive results. Also, socio-economic determinants have been drawn from these RDTs results which may have questionable implications. Thus, this study was aimed at evaluating the prevalence of malaria and hepatitis B by comparing RDTs with polymerase chain reaction (PCR) outcomes, and relating the PCR prevalence with socio-economic status among pregnant women in Northern Ghana.MethodsWe screened 2071 pregnant women on their first antenatal visit for Plasmodium falciparum and hepatitis B virus (HBV) using HRP2 and HBsAg RDTs, and confirming the infections with PCR. Socio-economic and obstetric information were collected using a pre-tested questionnaire, and associations with the infections were determined using Pearson's chi-square and multinomial logistic regression analyses at a significance level of pResultsThe prevalence of the infections by RDTs/PCR was: 14.1%/13.4% for P. falciparum mono-infection, 7.9%/7.5% for HBV mono-infection, and 1.9%/1.7% for P. falciparum/HBV co-infection. No statistical difference in prevalence rates were observed between the RDTs and PCRs (χ2  =  0.119, p = 0.73 for malaria and χ2  =  0.139, p = 0.709 for hepatitis B). Compared with PCRs, the sensitivity/specificity of the RDTs was 97.5%/99.1% and 97.9%/99.4% for HRP2 and HBsAg respectively. Socio-economic status was observed not to influence HBV mono-infection among the pregnant women (educational status: AOR = 0.78, 95% CI = 0.52-1.16, p = 0.222; economic status: AOR = 1.07, 95% CI = 0.72-1.56, p = 0.739; financial status: AOR = 0.66, 95% CI = 0.44-1.00, p = 0.052). However, pregnant women with formal education were at a lower risk for P. falciparum mono-infection (AOR = 0.48, 95% CI  =  0.32-0.71, pConclusionOur data has shown that, the RDTs are comparable to PCR and can give a representative picture of the prevalence of malaria and hepatitis B in endemic countries. Also, our results support the facts that improving socio-economic status is paramount in eliminating malaria in endemic settings. However, socio-economic status did not influence the prevalence of HBV mono-infection among pregnant women in Northern Ghana

A longitudinal two-year survey of the prevalence of trypanosomes in domestic cattle in Ghana by massively parallel sequencing of barcoded amplicons.

Funder: Cambridge-Africa Alborada Research FundBACKGROUND: Animal African Trypanosomiasis (AAT) is one of the most economically important diseases affecting livestock productivity in sub-Saharan Africa. The disease is caused by a broad range of Trypanosoma spp., infecting both wild and domesticated animals through cyclical and mechanical transmission. This study aimed to characterize trypanosomes present in cattle at regular intervals over two years in an AAT endemic and a non-endemic region of Ghana. METHODOLOGY/PRINCIPAL FINDINGS: Groups of cattle at Accra and Adidome were selected based on their geographical location, tsetse fly density, prevalence of trypanosomiasis and the breed of cattle available. Blood for DNA extraction was collected at approximately four to five-week intervals over a two-year period. Trypanosome DNA were detected by a sensitive nested PCR targeting the tubulin gene array and massively parallel sequencing of barcoded amplicons. Analysis of the data was a semi-quantitative estimation of infection levels using read counts obtained from the sequencing as a proxy for infection levels. Majority of the cattle were infected with multiple species most of the time [190/259 (73%) at Adidome and 191/324 (59%) at Accra], with T. vivax being the most abundant. The level of infection and in particular T. vivax, was higher in Adidome, the location with a high density of tsetse flies. The infection level varied over the time course, the timings of this variation were not consistent and in Adidome it appeared to be independent of prophylactic treatment for trypanosome infection. Effect of gender or breed on infection levels was insignificant. CONCLUSIONS/SIGNIFICANCE: Most cattle were infected with low levels of several trypanosome species at both study sites, with T. vivax being the most abundant. The measurements of infection over time provided insight to the importance of the approach in identifying cattle that could suppress trypanosome infection over an extended time and may serve as reservoir