Endemic Burkitt lymphoma avatar mouse models for exploring inter-patient tumor variation and testing targeted therapies.

Endemic Burkitt lymphoma (BL) is a childhood cancer in sub-Saharan Africa characterized by Epstein-Barr virus and malaria-associated aberrant B-cell activation and MYC chromosomal translocation. Survival rates hover at 50% after conventional chemotherapies; therefore, clinically relevant models are necessary to test additional therapies. Hence, we established five patient-derived BL tumor cell lines and corresponding NSG-BL avatar mouse models. Transcriptomics confirmed that our BL lines maintained fidelity from patient tumors to NSG-BL tumors. However, we found significant variation in tumor growth and survival among NSG-BL avatars and in Epstein-Barr virus protein expression patterns. We tested rituximab responsiveness and found one NSG-BL model exhibiting direct sensitivity, characterized by apoptotic gene expression counterbalanced by unfolded protein response and mTOR pro-survival pathways. In rituximab-unresponsive tumors, we observed an IFN-α signature confirmed by the expression of IRF7 and ISG15. Our results demonstrate significant inter-patient tumor variation and heterogeneity, and that contemporary patient-derived BL cell lines and NSG-BL avatars are feasible tools to guide new therapeutic strategies and improve outcomes for these children.

Interplay between IL-10, IFN-γ, IL-17A and PD-1 Expressing EBNA1-Specific CD4+ and CD8+ T Cell Responses in the Etiologic Pathway to Endemic Burkitt Lymphoma.

Children diagnosed with endemic Burkitt lymphoma (eBL) are deficient in interferon-γ (IFN-γ) responses to Epstein-Barr Nuclear Antigen1 (EBNA1), the viral protein that defines the latency I pattern in this B cell tumor. However, the contributions of immune-regulatory cytokines and phenotypes of the EBNA1-specific T cells have not been characterized for eBL. Using a bespoke flow cytometry assay we measured intracellular IFN-γ, IL-10, IL-17A expression and phenotyped CD4+ and CD8+ T cell effector memory subsets specific to EBNA1 for eBL patients compared to two groups of healthy children with divergent malaria exposures. In response to EBNA1 and a malaria antigen (PfSEA-1A), the three study groups exhibited strikingly different cytokine expression and T cell memory profiles. EBNA1-specific IFN-γ-producing CD4+ T cell response rates were lowest in eBL (40%) compared to children with high malaria (84%) and low malaria (66%) exposures (p < 0.0001 and p = 0.0004, respectively). However, eBL patients did not differ in CD8+ T cell response rates or the magnitude of IFN-γ expression. In contrast, eBL children were more likely to have EBNA1-specific CD4+ T cells expressing IL-10, and less likely to have polyfunctional IFN-γ+IL-10+ CD4+ T cells (p = 0.02). They were also more likely to have IFN-γ+IL-17A+, IFN-γ+ and IL-17A+ CD8+ T cell subsets compared to healthy children. Cytokine-producing T cell subsets were predominantly CD45RA+CCR7+ TNAIVE-LIKE cells, yet PD-1, a marker of persistent activation/exhaustion, was more highly expressed by the central memory (TCM) and effector memory (TEM) T cell subsets. In summary, our study suggests that IL-10 mediated immune regulation and depletion of IFN-γ+ EBNA1-specific CD4+ T cells are complementary mechanisms that contribute to impaired T cell cytotoxicity in eBL pathogenesis.

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.

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.

Association of maternal KIR gene content polymorphisms with reduction in perinatal transmission of HIV-1.

The role of killer cell immunoglobulin-like receptors (KIRs) in the transmission of HIV-1 has not been extensively studied. Here, we investigated the association of KIR gene content polymorphisms with perinatal HIV-1 transmission. The KIR gene family comprising 16 genes was genotyped in 313 HIV-1 positive Kenyan mothers paired with their infants. Gene content polymorphisms were presented as presence of individual KIR genes, haplotypes, genotypes and KIR gene concordance. The genetic data were analyzed for associations with perinatal transmission of HIV. There was no association of infant KIR genes with perinatal HIV-1 transmission. After adjustment for gravidity, viral load, and CD4 cell count, there was evidence of an association between reduction in perinatal HIV-1 transmission and the maternal individual KIR genes KIR2DL2 (adjusted OR = 0.50; 95% CI: 0.24-1.02, P = 0.06), KIR2DL5 (adjusted OR = 0.47; 95% CI: 0.23-0.95, P = 0.04) and KIR2DS5 (adjusted OR = 0.39; 95% CI: 0.18-0.80, P = 0.01). Furthermore, these maternal KIR genes were only significantly associated with reduction in perinatal HIV transmission in women with CD4 cell count ≥ 350 cells/ µl and viral load <10000 copies/ml. Concordance analysis showed that when both mother and child had KIR2DS2, there was less likelihood of perinatal HIV-1 transmission (adjusted OR = 0.44; 95% CI: 0.20-0.96, P = 0.039). In conclusion, the maternal KIR genes KIR2DL2, KIR2DL5, KIR2DS5, and KIR2DS2 were associated with reduction of HIV-1 transmission from mother to child. Furthermore, maternal immune status is an important factor in the association of KIR with perinatal HIV transmission.

Lay health worker experiences administering a multi-level combination intervention to improve PMTCT retention.

BACKGROUND: The recent scale-up of prevention of mother-to-child transmission of HIV (PMTCT) services has rapidly accelerated antiretroviral therapy (ART) uptake among pregnant and postpartum women in sub-Saharan Africa. The Mother and Infant Retention for Health (MIR4Health) study evaluates the impact of a combination intervention administered by trained lay health workers to decrease attrition among HIV-positive women initiating PMTCT services and their infants through 6 months postpartum. METHODS: This was a qualitative study nested within the MIR4Health trial. MIR4Health was conducted at 10 health facilities in Nyanza, Kenya from September 2013 to September 2015. The trial intervention addressed behavioral, social, and structural barriers to PMTCT retention and included: appointment reminders via text and phone calls, follow-up and tracking for missed clinic visits, PMTCT health education at home visits and during clinic visits, and retention and adherence support and counseling. All interventions were administered by lay health workers. We describe results of a nested small qualitative inquiry which conducted two focus groups to assess the experiences and perceptions of lay health workers administering the interventions. Discussions were recorded and simultaneously transcribed and translated into English. Data were analyzed using framework analysis approach. RESULTS: Study findings show lay health workers played a critical role supporting mothers in PMTCT services across a range of behavioral, social, and structural domains, including improved communication and contact, health education, peer support, and patient advocacy and assistance. Findings also identified barriers to the uptake and implementation of the interventions, such as concerns about privacy and stigma, and the limitations of the healthcare system including healthcare worker attitudes. Overall, study findings indicate that lay health workers found the interventions to be feasible, acceptable, and well received by clients. CONCLUSIONS: Lay health workers played a fundamental role in supporting mothers engaged in PMTCT services and provided valuable feedback on the implementation of PMTCT interventions. Future interventions must include strategies to ensure client privacy, decrease stigma within communities, and address the practical limitations of health systems. This study adds important insight to the growing body of research on lay health worker experiences in HIV and PMTCT care. TRIAL REGISTRATION: Clinicaltrials.gov NCT01962220 .

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.

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.

Human and Epstein-Barr Virus miRNA Profiling as Predictive Biomarkers for Endemic Burkitt Lymphoma.

Endemic Burkitt lymphoma (eBL) is an aggressive B cell lymphoma and is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum malaria co-infections. Central to BL oncogenesis is the over-expression of the MYC proto-oncogene which is caused by a translocation of an Ig enhancer in approximation to the myc gene. While whole genome/transcriptome sequencing methods have been used to define driver mutations and transcriptional dysregulation, microRNA (miRNA) dysregulation and differential expression has yet to be fully characterized. We hypothesized that both human and EBV miRNAs contribute to eBL clinical presentation, disease progression, and poor outcomes. Using sensitive and precise deep sequencing, we identified miRNAs from 17 Kenyan eBL patient tumor samples and delineated the complement of both host and EBV miRNAs. One human miRNA, hsa-miR-10a-5p was found to be differentially expressed (DE), being down-regulated in jaw tumors relative to abdominal and in non-survivors compared to survivors. We also examined EBV miRNAs, which made up 2.7% of the miRNA composition in the eBL samples. However, we did not find any significant associations regarding initial patient outcome or anatomical presentation. Gene ontology analysis and pathway enrichment of previously validated targets of miR-10a-5p suggest that it can promote tumor cell survival as well as aid in evasion of apoptosis. To examine miR-10a-5p regulatory effect on gene expression in eBL, we performed a pairwise correlation coefficient analysis on the expression levels of all its validated targets. We found a significant enrichment of correlated target genes consistent with miR-10a-5p impacting expression. The functions of genes and their correlation fit with multiple target genes impacting tumor resilience. The observed downregulation of miR-10a and associated genes suggests a role for miRNA in eBL patient outcomes and has potential as a predictive biomarker that warrants further investigation.

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.

Untangling the Relationship Between Antiretroviral Therapy Use and Incident Pregnancy: A Marginal Structural Model Analysis Using Data From 47,313 HIV-Positive Women in East Africa.

BACKGROUND: Scale-up of triple-drug antiretroviral therapy (ART) in Africa has transformed the context of childbearing for HIV-positive women and may impact pregnancy incidence in HIV programs. METHODS: Using observational data from 47,313 HIV-positive women enrolled at 26 HIV clinics in Kenya and Uganda between 2001 and 2009, we calculated the crude cumulative incidence of pregnancy for the pre-ART and on-ART periods. The causal effect of ART use on incident pregnancy was assessed using inverse probability weighted marginal structural models, and the relationship was further explored in multivariable Cox models. RESULTS: Crude cumulative pregnancy incidence at 1 year after enrollment/ART initiation was 4.0% and 3.9% during the pre-ART and on-ART periods, respectively. In marginal structural models, ART use was not significantly associated with incident pregnancy [hazard ratio = 1.06; 95% confidence interval (CI): 0.99 to 1.12]. Similarly, in Cox models, there was no significant relationship between ART use and incident pregnancy (cause-specific hazard ratio: 0.98; 95% CI: 0.91 to 1.05), but effect modification was observed. Specifically, women who were pregnant at enrollment and on ART had an increased risk of incident pregnancy compared to those not pregnant at enrollment and not on ART (cause-specific hazard ratio: 1.11; 95% CI: 1.01 to 1.23). CONCLUSIONS: In this large cohort, ART initiation was not associated with incident pregnancy in the general population of women enrolling in HIV care but rather only among those pregnant at enrollment. This finding further highlights the importance of scaling up access to lifelong treatment for pregnant women.

Anaemia in HIV-infected pregnant women receiving triple antiretroviral combination therapy for prevention of mother-to-child transmission: a secondary analysis of the Kisumu breastfeeding study (KiBS).

OBJECTIVE: The prevalence of anaemia during pregnancy is estimated to be 35-75% in sub-Saharan Africa and is associated with an increased risk of maternal mortality. We evaluated the frequency and factors associated with anaemia in HIV-infected women undergoing antiretroviral (ARV) therapy for prevention of mother-to-child transmission (PMTCT) enrolled in The Kisumu Breastfeeding Study 2003-2009. METHODS: Maternal haematological parameters were monitored from 32 to 34 weeks of gestation to 2 years post-delivery among 522 enrolled women. Clinical and laboratory assessments for causes of anaemia were performed, and appropriate management was initiated. Anaemia was graded using the National Institutes of Health Division of AIDS 1994 Adult Toxicity Tables. Data were analysed using SAS software, v 9.2. The Wilcoxon two-sample rank test was used to compare groups. A logistic regression model was fitted to describe the trend in anaemia over time. RESULTS: At enrolment, the prevalence of any grade anaemia (Hb < 9.4 g/dl) was 61.8%, but fell during ARV therapy, reaching a nadir (7.4%) by 6 months post-partum. A total of 41 women (8%) developed severe anaemia (Hb < 7 g/dl) during follow-up; 2 (4.9%) were hospitalised for blood transfusion, whereas 3 (7.3%) were transfused while hospitalised (for delivery). The greatest proportion of severe anaemia events occurred around delivery (48.8%; n = 20). Anaemia (Hb ≥ 7 and < 9.4 g/dl) at enrolment was associated with severe anaemia at delivery (OR 5.87; 95% CI: 4.48, 7.68, P < 0.01). Few cases of severe anaemia coincided with clinical malaria (24.4%; n = 10) and helminth (7.3%; n = 3) infections. CONCLUSION: Resolution of anaemia among most participants during study follow-up was likely related to receipt of ARV therapy. Efforts should be geared towards addressing common causes of anaemia in HIV-infected pregnant women, prioritising initiation of ARV therapy and management of peripartum blood loss.

Anemia and Red Blood Cell Abnormalities in HIV-Infected and HIV-Exposed Breastfed Infants: A Secondary Analysis of the Kisumu Breastfeeding Study.

BACKGROUND: Anemia results in increased morbidity and mortality, underscoring the need to better understand its pathophysiology amongst HIV-exposed and infected children in sub-Saharan Africa, the region where most infant HIV exposure and infections occur. METHODS: This analysis used samples obtained from children in the Kisumu Breastfeeding Study (KiBS). KiBS was a longitudinal phase IIB, open-label, one-arm clinical trial, designed to investigate the safety, tolerability and effectiveness of a maternal triple-antiretroviral (ARV) regimen for prevention of mother-to-child transmission (PMTCT) of HIV, during late pregnancy and early infancy while breastfeeding. Blood samples from 482 children were obtained at birth, 2, 6, 10 and 14 weeks and 6, 9, 12, 18 and 24 months. Severity of anemia was graded using the NIH Division of AIDS (DAIDS) toxicity tables. We describe the proportion of children with anemia and anomalies in red blood cell parameters at various time points over 24 months and compare rates of anemia between HIV-infected and HIV-uninfected children and by mothers' ARV regimen and infant malaria infection. RESULTS: The proportion of children with anemia significantly increased after the breastfeeding period in both HIV-infected and HIV-uninfected children with higher proportion among HIV-infected children compared to HIV-uninfected children (RR: 1.72; CI: 1.22-2.44, p = 0.002). Maternal triple-antiretroviral regimen was not associated with infant anemia (p = 0.11). There was no significant difference in mean hemoglobin between HIV-uninfected children with and without malaria at each time point except at 24 months. CONCLUSION: A relatively lower proportion of children with severe anemia during the breastfeeding period suggest that exposure to mother's triple antiretroviral combinations through breast milk, posed minimal risk of hematologic toxicity.

Incidence and predictors of first line antiretroviral regimen modification in western Kenya.

BACKGROUND: Limited antiretroviral treatment regimens in resource-limited settings require long-term sustainability of patients on the few available options. We evaluated the incidence and predictors of combined antiretroviral treatment (cART) modifications, in an outpatient cohort of 955 patients who initiated cART between January 2009 and January 2011 in western Kenya. METHODS: cART modification was defined as either first time single drug substitution or switch. Incidence rates were determined by Poisson regression and risk factor analysis assessed using multivariate Cox regression modeling. RESULTS: Over a median follow-up period of 10.7 months, 178 (18.7%) patients modified regimens (incidence rate (IR); 18.6 per 100 person years [95% CI: 16.2-21.8]). Toxicity was the most common cited reason (66.3%). In adjusted multivariate Cox piecewise regression model, WHO disease stage III/IV (aHR; 1.82, 95%CI: 1.25-2.66), stavudine (d4T) use (aHR; 2.21 95%CI: 1.49-3.30) and increase in age (aHR; 1.02, 95%CI: 1.0-1.04) were associated with increased risk of treatment modification within the first year post-cART. Zidovudine (AZT) and tenofovir (TDF) use had a reduced risk for modification (aHR; 0.60 95%CI: 0.38-0.96 and aHR; 0.51 95%CI: 0.29-0.91 respectively). Beyond one year of treatment, d4T use (aHR; 2.75, 95% CI: 1.25-6.05), baseline CD4 counts ≤350 cells/mm3 (aHR; 2.45, 95%CI: 1.14-5.26), increase in age (aHR; 1.05 95%CI: 1.02-1.07) and high baseline weight >60kg aHR; 2.69 95% CI: 1.58-4.59) were associated with risk of cART modification. CONCLUSIONS: Early treatment initiation at higher CD4 counts and avoiding d4T use may reduce treatment modification and subsequently improve sustainability of patients on the available limited options.

Sickle cell trait is not associated with endemic Burkitt lymphoma: an ethnicity and malaria endemicity-matched case-control study suggests factors controlling EBV may serve as a predictive biomarker for this pediatric cancer.

Endemic Burkitt lymphoma (eBL) is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum coinfections. Malaria appears to dysregulate immunity that would otherwise control EBV, thereby contributing to eBL etiology. Juxtaposed to human genetic variants associated with protection from malaria, it has been hypothesized that such variants could decrease eBL susceptibility, historically referred to as "the protective hypothesis." Past studies attempting to link sickle cell trait (HbAS), which is known to be protective against malaria, with protection from eBL were contradictory and underpowered. Therefore, using a case-control study design, we examined HbAS frequency in 306 Kenyan children diagnosed with eBL compared to 537 geographically defined and ethnically matched controls. We found 23.8% HbAS for eBL patients, which was not significantly different compared to 27.0% HbAS for controls [odds ratio (OR) = 0.85; 95% confidence interval (CI) 0.61-1.17; p-value = 0.33]. Even though cellular EBV titers, indicative of the number of latently infected B cells, were significantly higher (p-value < 0.0003) in children residing in malaria holoendemic compared to hypoendemic areas, levels were not associated with HbAS genotype. Combined, this suggests that although HbAS protects against severe malaria and hyperparasitemia, it is not associated with viral control or eBL protection. However, based on receiver operating characteristic curves factors that enable the establishment of EBV persistence, in contrast to those involved in EBV lytic reactivation, may have utility as an eBL precursor biomarker. This has implications for future human genetic association studies to consider variants influencing control over EBV in addition to malaria as risk factors for eBL.

Factors influencing time to diagnosis and initiation of treatment of endemic Burkitt Lymphoma among children in Uganda and western Kenya: a cross-sectional survey.

BACKGROUND: Survival rates for children diagnosed with Burkitt lymphoma (BL) in Africa are far below those achieved in developed countries. Late stage of presentation contributes to poor prognosis, therefore this study investigated factors leading to delays in BL diagnosis and treatment of children in Uganda and western Kenya. METHODS: Guardians of children diagnosed with BL were interviewed at the Jaramogi Oginga Odinga Teaching and Referral Hospital (JTRH) and Uganda Cancer Institute (UCI) from Jan-Dec 2010. Information on sociodemographics, knowledge, attitudes, illness perceptions, health-seeking behaviors and prior health encounters was collected using a standardized, pre-tested questionnaire. RESULTS: Eighty-two guardians were interviewed (20 JTRH, 62 UCI). Median "total delay" (1st symptoms to BL diagnosis) was 12.1 weeks [interquartile range (IQR) 4.9-19.9] in Kenya and 12.9 weeks (IQR 4.3-25.7) in Uganda. In Kenya, median "guardian delay" (1st symptoms to 1st health encounter) and "health system delay" (1st health encounter to BL diagnosis) were 9.0 weeks (IQR 3.6-15.7) and 2.0 weeks (IQR 1.6-5.8), respectively. Data on guardian and health system delay in Uganda were only available for those with < 4 prior health encounters (n = 26). Of these, median guardian delay was 4.3 weeks (range 0.7-149.9), health system delay 2.6 weeks (range 0.1-16.0), and total delay 10.7 weeks (range 1.7-154.3). Guardians in Uganda reported more health encounters than those in Kenya (median 5, range 3-16 vs. median 3, range 2-6). Among Kenyan guardians, source of income was the only independent predictor of delay, whereas in Uganda, guardian delay was influenced by guardians' beliefs on the curability of cancer, health system delay, by guardians' perceptions of cancer as a contagious disease, and total delay, by the number of children in the household and guardians' role as caretaker. Qualitative findings suggest financial costs, transportation, and other household responsibilities were major barriers to care. CONCLUSIONS: Delays from symptom onset to BL treatment were considerable given the rapid growth rate of this cancer, with guardian delay constituting the majority of total delay in both settings. Future interventions should aim to reduce structural barriers to care and increase awareness of BL in particular and cancer in general within the community, as well as among health professionals.

Differential association of gene content polymorphisms of killer cell immunoglobulin-like receptors with placental malaria in HIV- and HIV+ mothers.

Pregnant women have abundant natural killer (NK) cells in their placenta, and NK cell function is regulated by polymorphisms of killer cell immunoglobulin-like receptors (KIRs). Previous studies report different roles of NK cells in the immune responses to placental malaria (PM) and human immunodeficiency virus (HIV-1) infections. Given these references, the aim of this study was to determine the association between KIR gene content polymorphism and PM infection in pregnant women of known HIV-1 status. Sixteen genes in the KIR family were analyzed in 688 pregnant Kenyan women. Gene content polymorphisms were assessed in relation to PM in HIV-1 negative and HIV-1 positive women, respectively. Results showed that in HIV-1 negative women, the presence of the individual genes KIR2DL1 and KIR2DL3 increased the odds of having PM, and the KIR2DL2/KIR2DL2 homozygotes were associated with protection from PM. However, the reverse relationship was observed in HIV-1 positive women, where the presence of individual KIR2DL3 was associated with protection from PM, and KIR2DL2/KIR2DL2 homozygotes increased the odds for susceptibility to PM. Further analysis of the HIV-1 positive women stratified by CD4 counts showed that this reverse association between KIR genes and PM remained only in the individuals with high CD4 cell counts but not in those with low CD4 cell counts. Collectively, these results suggest that inhibitory KIR2DL2 and KIR2DL3, which are alleles of the same locus, play a role in the inverse effects on PM and PM/HIV co-infection and the effect of KIR genes on PM in HIV positive women is dependent on high CD4 cell counts. In addition, analysis of linkage disequilibrium (LD) of the PM relevant KIR genes showed strong LD in women without PM regardless of their HIV status while LD was broken in those with PM, indicating possible selection pressure by malaria infection on the KIR genes.

Trends in the clinical characteristics of HIV-infected patients initiating antiretroviral therapy in Kenya, Uganda and Tanzania between 2002 and 2009.

BACKGROUND: East Africa has experienced a rapid expansion in access to antiretroviral therapy (ART) for HIV-infected patients. Regionally representative socio-demographic, laboratory and clinical characteristics of patients accessing ART over time and across sites have not been well described. METHODS: We conducted a cross-sectional analysis of characteristics of HIV-infected adults initiating ART between 2002 and 2009 in Kenya, Uganda and Tanzania and in the International Epidemiologic Databases to Evaluate AIDS Consortium. Characteristics associated with advanced disease (defined as either a CD4 cell count level of less than 50 cells/mm3 or a WHO Stage 4 condition) at the time of ART initiation and use of stavudine (D4T) or nevirapine (NVP) were identified using a log-link Poisson model with robust standard errors. RESULTS: Among 48,658 patients (69% from Kenya, 22% from Uganda and 9% from Tanzania) accessing ART at 30 clinic sites, the median age at the time of ART initiation was 37 years (IQR: 31-43) and 65% were women. Pre-therapy CD4 counts rose from 87 cells/mm3 (IQR: 26-161) in 2002-03 to 154 cells/mm3 (IQR: 71-233) in 2008-09 (p<0.001). Accessing ART at advanced disease peaked at 35% in 2005-06 and fell to 27% in 2008-09. D4T use in the initial regimen fell from a peak of 88% in 2004-05 to 59% in 2008-09, and a greater extent of decline was observed in Uganda than in Kenya and Tanzania. Self-pay for ART peaked at 18% in 2003, but fell to less than 1% by 2005. In multivariable analyses, accessing ART at advanced immunosuppression was associated with male sex, women without a history of treatment for prevention of mother to child transmission (both as compared with women with such a history) and younger age after adjusting for year of ART initiation and country of residence. Receipt of D4T in the initial regimen was associated with female sex, earlier year of ART initiation, higher WHO stage, and lower CD4 levels at ART initiation and the absence of co-prevalent tuberculosis. CONCLUSIONS: Public health ART services in east Africa have improved over time, but the fraction of patients accessing ART with advanced immunosuppression is still high, men consistently access ART with more advanced disease, and D4T continues to be common in most settings. Strategies to facilitate access to ART, overcome barriers among men and reduce D4T use are needed.