Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania, and continued local transmission. To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo District on the coastal mainland from 2016-2018. Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Our data support importation as a main source of genetic diversity and contribution to the parasite population on Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive for malaria reemergence due to susceptible hosts and competent vectors.

Viral and host factors drive a type 1 Epstein-Barr virus spontaneous lytic phenotype.

IMPORTANCE: Epstein-Barr virus (EBV) infects over 95% of adults worldwide. Given its connection to various cancers and autoimmune disorders, it is important to understand the mechanisms by which infection with EBV can lead to these diseases. In this study, we describe an unusual spontaneous lytic phenotype in EBV strains isolated from Kenyan endemic Burkitt lymphoma patients. Because lytic replication of EBV has been linked to the pathogenesis of various diseases, these data could illuminate viral and host factors involved in this process.

Profiling genome-wide recombination in Epstein Barr virus reveals type-specific patterns and associations with endemic-Burkitt lymphoma.

BACKGROUND: Endemic Burkitt lymphoma (eBL) is potentiated through the interplay of Epstein Barr virus (EBV) and holoendemic Plasmodium falciparum malaria. To better understand EBV's biology and role in eBL, we characterized genome-wide recombination sites and patterns as a source of genetic diversity in EBV genomes in our well-defined population of eBL cases and controls from Western Kenya. METHODS: EBV genomes representing 54 eBL cases and 32 healthy children from the same geographic region in Western Kenya that we previously sequenced were analyzed. Whole-genome multiple sequence alignment, recombination analyses, and phylogenetic inference were made using multiple alignment with fast Fourier transform, recombination detection program 4, and molecular evolutionary genetics analysis. RESULTS: We identified 28 different recombination events and 71 (82.6%) of the 86 EBV genomes analyzed contained evidence of one or more recombinant segments. Associated recombination breakpoints were found to occur in a total of 42 different genes, with only 7 (16.67%) being latent genes. Recombination events were major drivers of clustering within genome-wide phylogenetic trees. The occurrence of recombination segments was comparable between genomes from male and female participants and across age groups. More recombinant segments were found in EBV type 1 genomes (p = 6.4e - 06) and the genomes from the eBLs (p = 0.037). Two recombination events were enriched in the eBLs; event 47 (OR = 4.07, p = 0.038) and event 50 (OR = 14.24, p = 0.012). CONCLUSIONS: EBV genomes have extensive evidence of recombination likely acquired progressively and cumulatively over time. Recombination patterns display a heterogeneous occurrence rate across the genome with enrichment in lytic genes. Overall, recombination appears to be a major evolutionary force impacting EBV diversity and genome structure with evidence of the association of specific recombinants with eBL.

Pediatric Participant Retention Rates in a Longitudinal Malaria Immunology Study.

The resurgence of drug-resistant Plasmodium falciparum parasites continues to motivate the development of a safe and efficacious malaria vaccine. Immuno-epidemiologic studies of naturally acquired immunity (NAI) have been a useful strategy to identify new malaria vaccine targets. However, retention of pediatric participants throughout longitudinal studies is essential for gathering comprehensive exposure and outcome data. Within the context of a 3-year cohort (N = 400) study involving monthly finger prick and bi-annual venous blood sample collections, we conducted qualitative surveys to assess factors impacting participant retention. Phase 1 was conducted 3 months after enrollment in July 2018 and phase 2, 12 months later. In phase 1, 236 parents/guardians participated in focus groups and three withdrawn participants and 10 community health volunteers (CHVs) in key informant interviews. Qualitative analysis indicated overall satisfaction with the study, with 61.8% (136/220 respondents) reporting no concerns. Focus group discussants associated attendance with benefits such as improved access to comprehensive healthcare services. Community health volunteers reported concerns over village rumors of inappropriate use of blood samples and dangers associated with venous blood draws. Phase 2 involved 205 parents/guardians and revealed continued satisfaction, with 46.3% (95/205) identifying no concerns, but expressed increasing worries regarding the amount of venous blood sample. This concern was reflected in an uptick of missed visits when venous blood samples were scheduled. Future studies will address parental concerns to determine whether community engagement and education measures increase study retention until completion.

Field evaluation of the diagnostic performance of EasyScan GO: a digital malaria microscopy device based on machine-learning.

BACKGROUND: Microscopic examination of Giemsa-stained blood films remains the reference standard for malaria parasite detection and quantification, but is undermined by difficulties in ensuring high-quality manual reading and inter-reader reliability. Automated parasite detection and quantification may address this issue. METHODS: A multi-centre, observational study was conducted during 2018 and 2019 at 11 sites to assess the performance of the EasyScan Go, a microscopy device employing machine-learning-based image analysis. Sensitivity, specificity, accuracy of species detection and parasite density estimation were assessed with expert microscopy as the reference. Intra- and inter-device reliability of the device was also evaluated by comparing results from repeat reads on the same and two different devices. This study has been reported in accordance with the Standards for Reporting Diagnostic accuracy studies (STARD) checklist. RESULTS: In total, 2250 Giemsa-stained blood films were prepared and read independently by expert microscopists and the EasyScan Go device. The diagnostic sensitivity of EasyScan Go was 91.1% (95% CI 88.9-92.7), and specificity 75.6% (95% CI 73.1-78.0). With good quality slides sensitivity was similar (89.1%, 95%CI 86.2-91.5), but specificity increased to 85.1% (95%CI 82.6-87.4). Sensitivity increased with parasitaemia rising from 57% at < 200 parasite/µL, to ≥ 90% at > 200-200,000 parasite/µL. Species were identified accurately in 93% of Plasmodium falciparum samples (kappa = 0.76, 95% CI 0.69-0.83), and in 92% of Plasmodium vivax samples (kappa = 0.73, 95% CI 0.66-0.80). Parasite density estimates by the EasyScan Go were within ± 25% of the microscopic reference counts in 23% of slides. CONCLUSIONS: The performance of the EasyScan Go in parasite detection and species identification accuracy fulfil WHO-TDR Research Malaria Microscopy competence level 2 criteria. In terms of parasite quantification and false positive rate, it meets the level 4 WHO-TDR Research Malaria Microscopy criteria. All performance parameters were significantly affected by slide quality. Further software improvement is required to improve sensitivity at low parasitaemia and parasite density estimations. Trial registration ClinicalTrials.gov number NCT03512678.

Low Complexity of Infection Is Associated With Molecular Persistence of Plasmodium falciparum in Kenya and Tanzania.

Background: Plasmodium falciparum resistance to artemisinin-based combination therapies (ACTs) is a threat to malaria elimination. ACT-resistance in Asia raises concerns for emergence of resistance in Africa. While most data show high efficacy of ACT regimens in Africa, there have been reports describing declining efficacy, as measured by both clinical failure and prolonged parasite clearance times. Methods: Three hundred children aged 2-10 years with uncomplicated P. falciparum infection were enrolled in Kenya and Tanzania after receiving treatment with artemether-lumefantrine. Blood samples were taken at 0, 24, 48, and 72 h, and weekly thereafter until 28 days post-treatment. Parasite and host genetics were assessed, as well as clinical, behavioral, and environmental characteristics, and host anti-malarial serologic response. Results: While there was a broad range of clearance rates at both sites, 85% and 96% of Kenyan and Tanzanian samples, respectively, were qPCR-positive but microscopy-negative at 72 h post-treatment. A greater complexity of infection (COI) was negatively associated with qPCR-detectable parasitemia at 72 h (OR: 0.70, 95% CI: 0.53-0.94), and a greater baseline parasitemia was marginally associated with qPCR-detectable parasitemia (1,000 parasites/uL change, OR: 1.02, 95% CI: 1.01-1.03). Demographic, serological, and host genotyping characteristics showed no association with qPCR-detectable parasitemia at 72 h. Parasite haplotype-specific clearance slopes were grouped around the mean with no association detected between specific haplotypes and slower clearance rates. Conclusions: Identifying risk factors for slow clearing P. falciparum infections, such as COI, are essential for ongoing surveillance of ACT treatment failure in Kenya, Tanzania, and more broadly in sub-Saharan Africa.

Comparative genomic and phenotypic characterization of invasive non-typhoidal Salmonella isolates from Siaya, Kenya.

Non-typhoidal Salmonella (NTS) is a major global health concern that often causes bloodstream infections in areas of the world affected by malnutrition and comorbidities such as HIV and malaria. Developing a strategy to control the emergence and spread of highly invasive and antimicrobial resistant NTS isolates requires a comprehensive analysis of epidemiological factors and molecular pathogenesis. Here, we characterize 11 NTS isolates that caused bloodstream infections in pediatric patients in Siaya, Kenya from 2003-2010. Nine isolates were identified as S. Typhimurium sequence type 313 while the other two were S. Enteritidis. Comprehensive genotypic and phenotypic analyses were performed to compare these isolates to those previously identified in sub-Saharan Africa. We identified a S. Typhimurium isolate referred to as UGA14 that displayed novel plasmid, pseudogene and resistance features as compared to other isolates reported from Africa. Notably, UGA14 is able to ferment both lactose and sucrose due to the acquisition of insertion elements on the pKST313 plasmid. These findings show for the first time the co-evolution of plasmid-mediated lactose and sucrose metabolism along with cephalosporin resistance in NTS further elucidating the evolutionary mechanisms of invasive NTS phenotypes. These results further support the use of combined genomic and phenotypic approaches to detect and characterize atypical NTS isolates in order to advance biosurveillance efforts that inform countermeasures aimed at controlling invasive and antimicrobial resistant NTS.

Epstein-Barr Virus Genomes Reveal Population Structure and Type 1 Association with Endemic Burkitt Lymphoma.

Endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in sub-Saharan Africa, is distinguished by its inclusion of Epstein-Barr virus (EBV). In order to better understand the impact of EBV variation in eBL tumorigenesis, we improved viral DNA enrichment methods and generated a total of 98 new EBV genomes from both eBL cases (n = 58) and healthy controls (n = 40) residing in the same geographic region in Kenya. Using our unbiased methods, we found that EBV type 1 was significantly more prevalent in eBL patients (74.5%) than in healthy children (47.5%) (odds ratio = 3.24, 95% confidence interval = 1.36 to 7.71, P = 0.007), as opposed to similar proportions in both groups. Controlling for EBV type, we also performed a genome-wide association study identifying six nonsynonymous variants in the genes EBNA1, EBNA2, BcLF1, and BARF1 that were enriched in eBL patients. In addition, viruses isolated from plasma of eBL patients were identical to their tumor counterparts consistent with circulating viral DNA originating from the tumor. We also detected three intertypic recombinants carrying type 1 EBNA2 and type 2 EBNA3 regions, as well as one novel genome with a 20-kb deletion, resulting in the loss of multiple lytic and virion genes. Comparing EBV types, viral genes displayed differential variation rates as type 1 appeared to be more divergent, while type 2 demonstrated novel substructures. Overall, our findings highlight the complexities of the EBV population structure and provide new insight into viral variation, potentially deepening our understanding of eBL oncogenesis.IMPORTANCE Improved viral enrichment methods conclusively demonstrate EBV type 1 to be more prevalent in eBL patients than in geographically matched healthy controls, which previously underrepresented the prevalence of EBV type 2. Genome-wide association analysis between cases and controls identifies six eBL-associated nonsynonymous variants in EBNA1, EBNA2, BcLF1, and BARF1 genes. Analysis of population structure reveals that EBV type 2 exists as two genomic subgroups and was more commonly found in female than in male eBL patients.

Cyclooxygenase-2 haplotypes influence the longitudinal risk of malaria and severe malarial anemia in Kenyan children from a holoendemic transmission region.

Cyclooxygenase-2 [(COX-2) or prostaglandin endoperoxide H2 synthase-2 (PTGS-2)] induces the production of prostaglandins as part of the host-immune response to infections. Although a number of studies have demonstrated the effects of COX-2 promoter variants on autoimmune and inflammatory diseases, their role in malaria remains undefined. As such, we investigated the relationship between four COX-2 promoter variants (COX-2 -512 C > T, -608 T > C, -765 G > C, and -1195 A > G) and susceptibility to malaria and severe malarial anemia (SMA) upon enrollment and longitudinally over a 36-month follow-up period. All-cause mortality was also explored. The investigation was carried out in children (n = 1081, age; 2-70 months) residing in a holoendemic Plasmodium falciparum transmission region of western Kenya. At enrollment, genotypes/haplotypes (controlling for anemia-promoting covariates) did not reveal any strong effects on susceptibility to either malaria or SMA. Longitudinal analyses showed decreased malaria episodes in children who inherited the -608 CC mutant allele (RR = 0.746, P = 1.811 × 10-4) and -512C/-608T/-765G/-1195G (CTGG) haplotype (RR = 0.856, P = 0.011), and increased risk in TTCA haplotype carriers (RR = 1.115, P = 0.026). Over the follow-up period, inheritance of the rare TTCG haplotype was associated with enhanced susceptibility to both malaria (RR = 1.608, P = 0.016) and SMA (RR = 5.714, P = 0.004), while carriage of the rare TTGG haplotype increased the risk of malaria (RR = 1.755, P = 0.007), SMA (RR = 8.706, P = 3.97 × 10-4), and all-cause mortality (HR = 110.000, P = 0.001). Collectively, these results show that SNP variations in the COX-2 promoter, and their inherited combinations, are associated with the longitudinal risk of malaria, SMA, and all-cause mortality among children living in a high transmission area for P. falciparum.

The whole-genome landscape of Burkitt lymphoma subtypes.

Burkitt lymphoma (BL) is an aggressive, MYC-driven lymphoma comprising 3 distinct clinical subtypes: sporadic BLs that occur worldwide, endemic BLs that occur predominantly in sub-Saharan Africa, and immunodeficiency-associated BLs that occur primarily in the setting of HIV. In this study, we comprehensively delineated the genomic basis of BL through whole-genome sequencing (WGS) of 101 tumors representing all 3 subtypes of BL to identify 72 driver genes. These data were additionally informed by CRISPR screens in BL cell lines to functionally annotate the role of oncogenic drivers. Nearly every driver gene was found to have both coding and non-coding mutations, highlighting the importance of WGS for identifying driver events. Our data implicate coding and non-coding mutations in IGLL5, BACH2, SIN3A, and DNMT1. Epstein-Barr virus (EBV) infection was associated with higher mutation load, with type 1 EBV showing a higher mutational burden than type 2 EBV. Although sporadic and immunodeficiency-associated BLs had similar genetic profiles, endemic BLs manifested more frequent mutations in BCL7A and BCL6 and fewer genetic alterations in DNMT1, SNTB2, and CTCF. Silencing mutations in ID3 were a common feature of all 3 subtypes of BL. In vitro, mass spectrometry-based proteomics demonstrated that the ID3 protein binds primarily to TCF3 and TCF4. In vivo knockout of ID3 potentiated the effects of MYC, leading to rapid tumorigenesis and tumor phenotypes consistent with those observed in the human disease.

Poorly cytotoxic terminally differentiated CD56negCD16pos NK cells accumulate in Kenyan children with Burkitt lymphomas.

Natural killer (NK) cells are critical for restricting viral infections and mediating tumor immunosurveillance. Epstein-Barr virus (EBV) and Plasmodium falciparum malaria are known risk factors for endemic Burkitt lymphoma (eBL), the most common childhood cancer in equatorial Africa. To date, the composition and function of NK cells have not been evaluated in eBL etiology or pathogenesis. Therefore, using multiparameter flow cytometry and in vitro killing assays, we compared NK cells from healthy children and children diagnosed with eBL in Kenya. We defined 5 subsets based on CD56 and CD16 expression, including CD56negCD16pos We found that licensed and terminally differentiated perforin-expressing CD56negCD16pos NK cells accumulated in eBL children, particularly in those with high EBV loads (45.2%) compared with healthy children without (6.07%) or with (13.5%) malaria exposure (P = .0007 and .002, respectively). This progressive shift in NK cell proportions was concomitant with fewer CD56dimCD16pos cells. Despite high MIP-1ß expression, CD56negCD16pos NK cells had diminished cytotoxicity, with lower expression of activation markers NKp46, NKp30, and CD160 and the absence of TNF-α. Of note, the accumulation of poorly cytotoxic CD56negCD16pos NK cells resolved in long-term eBL survivors. Our study demonstrates impaired NK cell-mediated immunosurveillance in eBL patients but with the potential to restore a protective NK cell repertoire after cancer treatment. Characterizing NK cell dysfunction during coinfections with malaria and EBV has important implications for designing immunotherapies to improve outcomes for children diagnosed with eBL.

Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma.

BACKGROUND: Burkitt lymphoma (BL) is characterized by overexpression of the c-myc oncogene, which in the vast majority of cases is a consequence of an IGH/MYC translocation. While myc is the seminal event, BL is a complex amalgam of genetic and epigenetic changes causing dysregulation of both coding and non-coding transcripts. Emerging evidence suggest that abnormal modulation of mRNA transcription via miRNAs might be a significant factor in lymphomagenesis. However, the alterations in these miRNAs and their correlations to their putative mRNA targets have not been extensively studied relative to normal germinal center (GC) B cells. METHODS: Using more sensitive and specific transcriptome deep sequencing, we compared previously published small miRNA and long mRNA of a set of GC B cells and eBL tumors. MiRWalk2.0 was used to identify the validated target genes for the deregulated miRNAs, which would be important for understanding the regulatory networks associated with eBL development. RESULTS: We found 211 differentially expressed (DE) genes (79 upregulated and 132 downregulated) and 49 DE miRNAs (22 up-regulated and 27 down-regulated). Gene Set enrichment analysis identified the enrichment of a set of MYC regulated genes. Network propagation-based method and correlated miRNA-mRNA expression analysis identified dysregulated miRNAs, including miR-17~95 cluster members and their target genes, which have diverse oncogenic properties to be critical to eBL lymphomagenesis. Central to all these findings, we observed the downregulation of ATM and NLK genes, which represent important regulators in response to DNA damage in eBL tumor cells. These tumor suppressors were targeted by multiple upregulated miRNAs (miR-19b-3p, miR-26a-5p, miR-30b-5p, miR-92a-5p and miR-27b-3p) which could account for their aberrant expression in eBL. CONCLUSION: Combined loss of p53 induction and function due to miRNA-mediated regulation of ATM and NLK, together with the upregulation of TFAP4, may be a central role for human miRNAs in eBL oncogenesis. This facilitates survival of eBL tumor cells with the IGH/MYC chromosomal translocation and promotes MYC-induced cell cycle progression, initiating eBL lymphomagenesis. This characterization of miRNA-mRNA interactions in eBL relative to GC B cells provides new insights on miRNA-mediated transcript regulation in eBL, which are potentially useful for new improved therapeutic strategies.

High pathogen burden in childhood promotes the development of unconventional innate-like CD8+ T cells.

Cellular and humoral constituents of the immune system differ significantly between children and adults, yet very little is known about the impact of early-life pathogen exposure on this immunologic transition. We examined CD4+ and CD8+ T cell subsets defined by CCR7 and CD45RA expression in two longitudinal pediatric cohorts experiencing divergent levels of pathogen burden. Using multiparameter flow cytometry, along with serological, cytokine, and transcriptomic data, we show that cumulative pathogen burden promotes the development of atypical CD8dim T cells with an innate-like profile (Granzyme Bhi, IFNγlow, TNFαlow, PLFZhi, ID2hi, IKZF2hi) in contrast to age-matched children residing in a low pathogen-exposure area who display a more conventional CD8bright profile (IFNγ+, TNFα+, CCL4+). Furthermore, these unconventional T cells had stunted proliferation, distinct transcriptional programs, and impaired T cell receptor signaling and were enriched in hallmark TNFα, NF-κB, and IL-6 gene signaling pathways, reminiscent of NK cells and type-1 innate lymphoid cells. Our findings suggest that these unconventional CD8dim T cells arise in a very particular immunological context and may provide a deeper understanding of the heterogeneity in human immune responses.

Comprehensive Transcriptome and Mutational Profiling of Endemic Burkitt Lymphoma Reveals EBV Type-Specific Differences.

Endemic Burkitt lymphoma (eBL) is the most common pediatric cancer in malaria-endemic equatorial Africa and nearly always contains Epstein-Barr virus (EBV), unlike sporadic Burkitt lymphoma (sBL) that occurs with a lower incidence in developed countries. Given these differences and the variable clinical presentation and outcomes, we sought to further understand pathogenesis by investigating transcriptomes using RNA sequencing (RNAseq) from multiple primary eBL tumors compared with sBL tumors. Within eBL tumors, minimal expression differences were found based on: anatomical presentation site, in-hospital survival rates, and EBV genome type, suggesting that eBL tumors are homogeneous without marked subtypes. The outstanding difference detected using surrogate variable analysis was the significantly decreased expression of key genes in the immunoproteasome complex (PSMB9/ß1i, PSMB10/ß2i, PSMB8/ß5i, and PSME2/PA28ß) in eBL tumors carrying type 2 EBV compared with type 1 EBV. Second, in comparison with previously published pediatric sBL specimens, the majority of the expression and pathway differences was related to the PTEN/PI3K/mTOR signaling pathway and was correlated most strongly with EBV status rather than geographic designation. Third, common mutations were observed significantly less frequently in eBL tumors harboring EBV type 1, with mutation frequencies similar between tumors with EBV type 2 and without EBV. In addition to the previously reported genes, a set of new genes mutated in BL, including TFAP4, MSH6, PRRC2C, BCL7A, FOXO1, PLCG2, PRKDC, RAD50, and RPRD2, were identified. Overall, these data establish that EBV, particularly EBV type 1, supports BL oncogenesis, alleviating the need for certain driver mutations in the human genome. IMPLICATIONS: Genomic and mutational analyses of Burkitt lymphoma tumors identify key differences based on viral content and clinical outcomes suggesting new avenues for the development of prognostic molecular biomarkers and therapeutic interventions.

CD4 T-cell expression of IFN-γ and IL-17 in pediatric malarial anemia.

In Plasmodium falciparum holoendemic transmission regions of western Kenya, life-threatening pediatric malaria manifests primarily as severe malarial anemia (SMA, Hb≤6.0 g/dL with any density parasitemia). To determine the role that CD4+ T-cell-driven inflammatory responses have in the pathogenesis of SMA, peripheral CD4+ T-cell populations and their intracellular production of pro-inflammatory cytokines (IFN-γ and IL-17) were characterized in children aged 12-36 months of age stratified into two groups: non-severe malarial anemia (non-SMA, Hb≥6.0 g/dL, n = 50) and SMA (n = 39). In addition, circulating IFN-γ and IL-17 were measured as part of a Cytokine 25-plex Antibody Bead Kit, Human (BioSource™ International). Children with SMA had higher overall proportions of circulating lymphocytes (P = 0.003) and elevated proportions of lymphocytes expressing IFN-γ (P = 0.014) and comparable IL-17 (P = 0.101). In addition, SMA was characterized by decreased memory-like T-cells (CD4+CD45RA-) expressing IL-17 (P = 0.009) and lower mean fluorescence intensity in memory-like CD4+ T-cells for both IFN-γ (P = 0.063) and IL-17 (P = 0.006). Circulating concentrations of IFN-γ were higher in children with SMA (P = 0.009), while IL-17 levels were comparable between the groups (P = 0.164). Furthermore, circulating levels of IFN-γ were negatively correlated with IL-17 levels in both groups of children (SMA: r = -0.610, P = 0.007; and non-SMA: r = -0.516, P = 0.001), while production of both cytokines by lymphocytes were positively correlated (SMA: r = 0.349, P = 0.037; and non-SMA: r = 0.475, P = 0.001). In addition, this correlation was only maintained by the memory-like CD4+ T cells (r = 0.365, P = 0.002) but not the naïve-like CD4+ T cells. However, circulating levels of IFN-γ were only associated with naïve-like CD4+ T cells producing IFN-γ (r = 0.547, P = 0.028), while circulating levels of IL-17 were not associated with any of the cell populations. Taken together, these results suggest that enhanced severity of malarial anemia is associated with higher overall levels of circulating lymphocytes, enhanced intracellular production of IFN-γ by peripheral lymphocytes and high circulating IFN-γ levels. In addition, the observed inverse relationship between the circulating levels of IFN-γ and IL-17 together with the reduction in the levels of memory-like CD4+ T cells expressing IL-17 in children with SMA may suggest possible relocation of these cells in the deeper tissues for their pathological effect.

Regulatory T Cells in Endemic Burkitt Lymphoma Patients Are Associated with Poor Outcomes: A Prospective, Longitudinal Study.

Deficiencies in Epstein-Barr virus (EBV)-specific T cell immunosurveillance appear to precede the development of endemic Burkitt lymphoma (eBL), a malaria-associated pediatric cancer common in sub-Saharan Africa. However, T cell contributions to eBL disease progression and survival have not been characterized. Our objective was to investigate regulatory and inflammatory T cell responses in eBL patients associated with clinical outcomes. By multi-parameter flow cytometry, we examined peripheral blood mononuclear cells from 38 eBL patients enrolled in a prospective cohort study in Kisumu, Kenya from 2008-2010, and 14 healthy age-matched Kenyan controls. Children diagnosed with eBL were prospectively followed and outcomes categorized as 2-year event-free survivors, cases of relapses, or those who died. At the time of diagnosis, eBL children with higher CD25+Foxp3+ regulatory T (Treg) cell frequencies were less likely to survive than patients with lower Treg frequencies (p = 0·0194). Non-survivors also had higher absolute counts of CD45RA+Foxp3lo naïve and CD45RA-Foxp3hi effector Treg subsets compared to survivors and healthy controls. Once patients went into clinical remission, Treg frequencies remained low in event-free survivors. Patients who relapsed, however, showed elevated Treg frequencies months prior to their adverse event. Neither concurrent peripheral blood EBV load nor malaria infection could explain higher Treg cell frequencies. CD8+ T cell PD-1 expression was elevated in all eBL patients at time of diagnosis, but relapse patients tended to have persistently high PD-1 expression compared to long-term survivors. Non-survivors produced more CD4+ T-cell IL-10 in response to both Epstein-Barr Nuclear Antigen-1 (EBNA-1) (p = 0·026) and the malaria antigen Plasmodium falciparum Schizont Egress Antigen-1 (p = 0·0158) compared to survivors, and were concurrently deficient in (EBNA-1)-specific CD8+ T-cell derived IFN-γ production (p = 0·002). In addition, we identified the presence of Foxp3-IL10+ regulatory Type 1 cells responding to EBNA-1 in contrast to the malaria antigen tested. These novel findings suggest that poor outcomes in eBL patients are associated with a predominantly immuno-regulatory environment. Therefore, Treg frequencies could be a predictive biomarker of disease progression and manipulation of Treg activity has potential as a therapeutic target to improve eBL survival.

Factors influencing survival among Kenyan children diagnosed with endemic Burkitt lymphoma between 2003 and 2011: A historical cohort study.

Discovering how to improve survival and establishing clinical reference points for children diagnosed with endemic Burkitt lymphoma (eBL) in resource-constrained settings has recaptured international attention. Using multivariate analyses, we evaluated 428 children with eBL in Kenya for age, gender, tumor stage, nutritional status, hemoglobin, lactate dehydrogenase (LDH), Epstein-Barr virus (EBV) and Plasmodium falciparum prior to induction of chemotherapy (cyclophosphamide, vincristine, methotrexate and doxorubicin) to identify predictive and prognostic biomarkers of survival. During this 10 year prospective study period, 22% died in-hospital and 78% completed six-courses of chemotherapy. Of those, 16% relapsed or died later; 31% achieved event-free-survival; and 31% were lost to follow-up; the overall one-year survival was 45%. After adjusting for covariates, low hemoglobin (<8 g/dL) and high LDH (>400 mU/ml) were associated with increased risk of death (adjusted Hazard Ratio (aHR) = 1.57 [0.97-2.41]) and aHR = 1.84, [0.91-3.69], respectively). Anemic children with malaria were 3.55 times more likely to die [1.10-11.44] compared to patients without anemia or malarial infection. EBV load did not differ by tumor stage nor was it associated with survival. System-level factors can also contribute to poor outcomes. Children were more likely to die when inadvertently overdosed by more than 115% of the correct dose of cyclophosphamide (a HR = 1.43 [0.84-2.43]) or doxorubicin (a HR = 1.25, [0.66-2.35]), compared with those receiving accurate doses of the respective agent in this setting. This study codifies risk factors associated with poor outcomes for eBL patients in Africa and provides a benchmark by which to assess improvements in survival for new chemotherapeutic approaches.

Severe malarial anemia: innate immunity and pathogenesis.

Greater than 80% of malaria-related mortality occurs in sub-Saharan Africa due to infections with Plasmodium falciparum. The majority of P. falciparum-related mortality occurs in immune-naïve infants and young children, accounting for 18% of all deaths before five years of age. Clinical manifestations of severe falciparum malaria vary according to transmission intensity and typically present as one or more life-threatening complications, including: hyperparasitemia; hypoglycemia; cerebral malaria; severe malarial anemia (SMA); and respiratory distress. In holoendemic transmission areas, SMA is the primary clinical manifestation of severe childhood malaria, with cerebral malaria occurring only in rare cases. Mortality rates from SMA can exceed 30% in pediatric populations residing in holoendemic transmission areas. Since the vast majority of the morbidity and mortality occurs in immune-naïve African children less than five years of age, with SMA as the primary manifestation of severe disease, this review will focus primarily on the innate immune mechanisms that govern malaria pathogenesis in this group of individuals. The pathophysiological processes that contribute to SMA involve direct and indirect destruction of parasitized and non-parasitized red blood cells (RBCs), inefficient and/or suppression of erythropoiesis, and dyserythropoiesis. While all of these causal etiologies may contribute to reduced hemoglobin (Hb) concentrations in malaria-infected individuals, data from our laboratory and others suggest that SMA in immune-naïve children is characterized by a reduced erythropoietic response. One important cause of impaired erythroid responses in children with SMA is dysregulation in the innate immune response. Phagocytosis of malarial pigment hemozoin (Hz) by monocytes, macrophages, and neutrophils is a central factor for promoting dysregulation in innate inflammatory mediators. As such, the role of P. falciparum-derived Hz (PfHz) in mediating suppression of erythropoiesis through its ability to cause dysregulation in pro- and anti-inflammatory cytokines, growth factors, chemokines, and effector molecules is discussed in detail. An improved understanding of the etiological basis of suppression of erythropoietic responses in children with SMA may offer the much needed therapeutic alternatives for control of this global disease burden.

Acquisition of hemozoin by monocytes down-regulates interleukin-12 p40 (IL-12p40) transcripts and circulating IL-12p70 through an IL-10-dependent mechanism: in vivo and in vitro findings in severe malarial anemia.

Severe malarial anemia (SMA) is a primary cause of morbidity and mortality in immune-naïve infants and young children residing in areas of holoendemic Plasmodium falciparum transmission. Although the immunopathogenesis of SMA is largely undefined, we have previously shown that systemic interleukin-12 (IL-12) production is suppressed during childhood blood-stage malaria. Since IL-10 and tumor necrosis factor alpha (TNF-alpha) are known to decrease IL-12 synthesis in a number of infectious diseases, altered transcriptional regulation of these inflammatory mediators was investigated as a potential mechanism for IL-12 down-regulation. Ingestion of naturally acquired malarial pigment (hemozoin [PfHz]) by monocytes promoted the overproduction of IL-10 and TNF-alpha relative to the production of IL-12, which correlated with an enhanced severity of malarial anemia. Experiments with cultured peripheral blood mononuclear cells (PBMC) and CD14(+) cells from malaria-naïve donors revealed that physiological concentrations of PfHz suppressed IL-12 and augmented IL-10 and TNF-alpha by altering the transcriptional kinetics of IL-12p40, IL-10, and TNF-alpha, respectively. IL-10 neutralizing antibodies, but not TNF-alpha antibodies, restored PfHz-induced suppression of IL-12. Blockade of IL-10 and the addition of recombinant IL-10 to cultured PBMC from children with SMA confirmed that IL-10 was responsible for malaria-induced suppression of IL-12. Taken together, these results demonstrate that PfHz-induced up-regulation of IL-10 is responsible for the suppression of IL-12 during malaria.

Parasitemia, anemia, and malarial anemia in infants and young children in a rural holoendemic Plasmodium falciparum transmission area.

Malarial anemia (MA) is a multifactorial disease for which the complex etiological basis is only partially defined. The association of clinical, nutritional, demographic, and socioeconomic factors with parasitemia, anemia, and MA was determined for children presenting at a hospital in a holoendemic area of Plasmodium falciparum transmission in western Kenya. Parasitemia was not associated with malaria disease severity. In univariate logistic regression, fever was significantly associated with parasitemia, and wasting was associated with increased presentation of MA. Caretaker's level of education and occupation were significantly correlated with parasitemia, anemia, and MA. Housing structure was also significantly associated with parasitemia and anemia. Bed net use was protective against parasitemia but not anemia or MA. Multivariate logistic regression models demonstrated that fever, mother's occupation, and bed net use were associated with parasitemia. In the current study, none of the factors were associated with anemia or MA in the multivariate models.