Decrease in Numbers of Naive and Resting B Cells in HIV-Infected Kenyan Adults Leads to a Proportional Increase in Total and Plasmodium falciparum-Specific Atypical Memory B Cells.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with B cell activation and exhaustion, and hypergammaglobulinemia. How these changes influence B cell responses to coinfections such as malaria is poorly understood. To address this, we compared B cell phenotypes and Abs specific for the Plasmodium falciparum vaccine candidate apical membrane Ag-1 (AMA1) in HIV-infected and uninfected adults living in Kenya. Surprisingly, HIV-1 infection was not associated with a difference in serum AMA1-specific Ab levels. HIV-infected individuals had a higher proportion of total atypical and total activated memory B cells (MBCs). Using an AMA1 tetramer to detect AMA1-specific B cells, HIV-infected individuals were also shown to have a higher proportion of AMA1-specific atypical MBCs. However, this proportional increase resulted in large part from a loss in the number of naive and resting MBCs rather than an increase in the number of atypical and activated cells. The loss of resting MBCs and naive B cells was mirrored in a population of cells specific for an Ag to which these individuals were unlikely to have been chronically exposed. Together, the data show that changes in P. falciparum Ag-specific B cell subsets in HIV-infected individuals mirror those in the overall B cell population, and suggest that the increased proportion of atypical MBC phenotypes found in HIV-1-infected individuals results from the loss of naive and resting MBCs.

Use of menstrual cups among school girls: longitudinal observations nested in a randomised controlled feasibility study in rural western Kenya.

BACKGROUND: A menstrual cup can be a good solution for menstrual hygiene management in economically challenged settings. As part of a pilot study we assessed uptake and maintenance of cup use among young school girls in Kenya. METHODS: A total of 192 girls between 14 to 16 years were enrolled in 10 schools in Nyanza Province, Western Kenya; these schools were assigned menstrual cups as part of the cluster-randomized pilot study. Girls were provided with menstrual cups in addition to training and guidance on use, puberty education, and instructions for menstrual hygiene. During repeated individual visits with nurses, girls reported use of the menstrual cup and nurses recorded colour change of the cup. RESULTS: Girls were able to keep their cups in good condition, with only 12 cups (6.3%) lost (dropped in toilet, lost or destroyed). Verbally reported cup use increased from 84% in the first 3 months (n = 143) to 96% after 9 months (n = 74). Colour change of the cup, as 'uptake' indicator of use, was detected in 70.8% of 192 participants, with a median time of 5 months (range 1-14 months). Uptake differed by school and was significantly higher among girls who experienced menarche within the past year (adjusted risk ratio 1.29, 95% CI 1.04-1.60), and was faster among girls enrolled in the second study year (hazard ratio 3.93, 95% CI 2.09-7.38). The kappa score comparing self-report and cup colour observation was 0.044 (p = 0.028), indicating that agreement was only slightly higher than by random chance. CONCLUSIONS: Objective evidence through cup colour change suggests school girls in rural Africa can use menstrual cups, with uptake improving with peer group education and over time. TRIAL REGISTRATION: ISRCTN17486946 . Retrospectively registered 09 December 2014.

Unrecognized Dengue Virus Infections in Children, Western Kenya, 2014-2015.

We detected a cluster of dengue virus infections in children in Kenya during July 2014-June 2015. Most cases were serotype 1, but we detected all 4 serotypes, including co-infections with 2 serotypes. Our findings implicate dengue as a cause of febrile illness in this population and highlight a need for robust arbovirus surveillance.

Principles, practices and knowledge of clinicians when assessing febrile children: a qualitative study in Kenya.

BACKGROUND: Clinicians in low resource settings in malaria endemic regions face many challenges in diagnosing and treating febrile illnesses in children. Given the change in WHO guidelines in 2010 that recommend malaria testing prior to treatment, clinicians are now required to expand the differential when malaria testing is negative. Prior studies have indicated that resource availability, need for additional training in differentiating non-malarial illnesses, and lack of understanding within the community of when to seek care play a role in effective diagnosis and treatment. The objective of this study was to examine the various factors that influence clinician behavior in diagnosing and managing children presenting with fever to health centres in Kenya. METHODS: A total of 20 clinicians (2 paediatricians, 1 medical officer, 2 nurses, and 15 clinical officers) were interviewed, working at 5 different government-sponsored public clinic sites in two areas of Kenya where malaria is prevalent. Clinicians were interviewed one-on-one using a structured interview technique. Interviews were then analysed qualitatively for themes. RESULTS: The following five themes were identified: (1) Strong familiarity with diagnosis of malaria and testing for malaria; (2) Clinician concerns about community understanding of febrile illness, use of traditional medicine, delay in seeking care, and compliance; (3) Reliance on clinical guidelines, history, and physical examination to diagnose febrile illness and recognize danger signs; (4) Clinician discomfort with diagnosis of primary viral illness leading to increased use of empiric antibiotics; and (5) Lack of resources including diagnostic testing, necessary medications, and training modalities contributes to the difficulty clinicians face in assessing and treating febrile illness in children. These themes persisted across all sites, despite variation in levels of medical care. Within these themes, clinicians consistently expressed a need for reliable basic testing, especially haemograms and bacterial cultures. Clinicians discussed the use of counseling and education to improve community understanding of febrile illness in order to decrease preventable deaths in children. CONCLUSION: Results of this study suggest that since malarial testing has become more widespread, clinicians working in resource-poor environments still face difficulty when evaluating a child with fever, especially when malaria testing is negative. Improving access to additional diagnostics, continuing medical education, and ongoing evaluation and revision of clinical guidelines may lead to more consistent management of febrile illness by providers, and may potentially decrease prescription of unnecessary antibiotics. Additional interventions at the community level may also have an important role in managing febrile illness, however, more studies are needed to identify targets for intervention at both the clinic and community levels.

New gorilla adenovirus vaccine vectors induce potent immune responses and protection in a mouse malaria model.

BACKGROUND: A DNA-human Ad5 (HuAd5) prime-boost malaria vaccine has been shown to protect volunteers against a controlled human malaria infection. The potency of this vaccine, however, appeared to be affected by the presence of pre-existing immunity against the HuAd5 vector. Since HuAd5 seroprevalence is very high in malaria-endemic areas of the world, HuAd5 may not be the most appropriate malaria vaccine vector. This report describes the evaluation of the seroprevalence, immunogenicity and efficacy of three newly identified gorilla adenoviruses, GC44, GC45 and GC46, as potential malaria vaccine vectors. RESULTS: The seroprevalence of GC44, GC45 and GC46 is very low, and the three vectors are not efficiently neutralized by human sera from Kenya and Ghana, two countries where malaria is endemic. In mice, a single administration of GC44, GC45 and GC46 vectors expressing a murine malaria gene, Plasmodium yoelii circumsporozoite protein (PyCSP), induced robust PyCSP-specific T cell and antibody responses that were at least as high as a comparable HuAd5-PyCSP vector. Efficacy studies in a murine malaria model indicated that a prime-boost regimen with DNA-PyCSP and GC-PyCSP vectors can protect mice against a malaria challenge. Moreover, these studies indicated that a DNA-GC46-PyCSP vaccine regimen was significantly more efficacious than a DNA-HuAd5-PyCSP regimen. CONCLUSION: These data suggest that these gorilla-based adenovectors have key performance characteristics for an effective malaria vaccine. The superior performance of GC46 over HuAd5 highlights its potential for clinical development.

Pneumococcal carriage and antibiotic susceptibility patterns from two cross-sectional colonization surveys among children aged <5 years prior to the introduction of 10-valent pneumococcal conjugate vaccine - Kenya, 2009-2010.

BACKGROUND: Pneumococci are spread by persons with nasopharyngeal colonization, a necessary precursor to invasive disease. Pneumococcal conjugate vaccines can prevent colonization with vaccine serotype strains. In 2011, Kenya became one of the first African countries to introduce the 10-valent pneumococcal conjugate vaccine (PCV10) into its national immunization program. Serial cross-sectional colonization surveys were conducted to assess baseline pneumococcal colonization, antibiotic resistance patterns, and factors associated with resistance. METHODS: Annual surveys were conducted in one urban and one rural site during 2009 and 2010 among children aged <5 years. To reflect differences in vaccine target population, recruitment was age-stratified in Kibera, whereas a simple random sample of children was drawn in Lwak. Nasopharyngeal swabs were collected from eligible children. Pneumococci were isolated and serotyped. Antibiotic susceptibility testing was performed using the 2009 isolates. Antibiotic nonsusceptibility was defined as intermediate susceptibility or resistance to ≥1 antibiotics (i.e., penicillin, chloramphenicol, levofloxacin, erythromycin, tetracycline, cotrimoxazole, and clindamycin); multidrug resistance (MDR) was defined as nonsusceptibility to ≥3 antibiotics. Weighted analysis was conducted when appropriate. Modified Poisson regression was used to calculate factors associated with antibiotic nonsusceptibility. RESULTS: Of 1,087 enrolled (Kibera: 740, Lwak: 347), 90.0% of these were colonized with pneumococci, and 37.3% were colonized with PCV10 serotypes. There were no differences by survey site or year. Of 657 (of 730; 90%) isolates tested for antibiotic susceptibility, nonsusceptibility to cotrimoxazole and penicillin was found in 98.6 and 81.9% of isolates, respectively. MDR was found in 15.9% of isolates and most often involved nonsusceptibility to cotrimoxazole and penicillin; 40.4% of MDR isolates were PCV10 serotypes. In the multivariable model, PCV10 serotypes were independently associated with penicillin nonsusceptibility (Prevalence Ratio: 1.2, 95% CI 1.1-1.3), but not with MDR. CONCLUSIONS: Before PCV10 introduction, nearly all Kenyan children aged <5 years were colonized with pneumococci, and PCV10 serotype colonization was common. PCV10 serotypes were associated with penicillin nonsusceptibility. Given that colonization with PCV10 serotypes is associated with greater risk for invasive disease than colonization with other serotypes, successful PCV10 introduction in Kenya is likely to have a substantial impact in reducing vaccine-type pneumococcal disease and drug-resistant pneumococcal infection.

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.

Prevalence of reproductive tract infections and the predictive value of girls' symptom-based reporting: findings from a cross-sectional survey in rural western Kenya.

OBJECTIVES: Reproductive tract infections (RTIs), including sexually acquired, among adolescent girls is a public health concern, but few studies have measured prevalence in low-middle-income countries. The objective of this study was to examine prevalence in rural schoolgirls in Kenya against their reported symptoms. METHODS: In 2013, a survey was conducted in 542 adolescent schoolgirls aged 14-17 years who were enrolled in a menstrual feasibility study. Vaginal self-swabbing was conducted after girls were interviewed face-to-face by trained nurses on symptoms. The prevalence of girls with symptoms and laboratory-confirmed infections, and the sensitivity, specificity, positive and negative predictive values of symptoms compared with laboratory results, were calculated. RESULTS: Of 515 girls agreeing to self-swab, 510 answered symptom questions. A quarter (24%) reported one or more symptoms; most commonly vaginal discharge (11%), pain (9%) or itching (4%). Laboratory tests confirmed 28% of girls had one or more RTI. Prevalence rose with age; among girls aged 16-17 years, 33% had infections. Bacterial vaginosis was the most common (18%), followed by Candida albicans (9%), Chlamydia trachomatis (3%), Trichomonas vaginalis (3%) and Neisseria gonorrhoeae (1%). Reported symptoms had a low sensitivity and positive predictive value. Three-quarters of girls with bacterial vaginosis and C. albicans, and 50% with T. vaginalis were asymptomatic. CONCLUSIONS: There is a high prevalence of adolescent schoolgirls with RTI in rural Kenya. Public efforts are required to identify and treat infections among girls to reduce longer-term sequelae but poor reliability of symptom reporting minimises utility of symptom-based diagnosis in this population. TRIAL REGISTRATION NUMBER: ISRCTN17486946.

High Streptococcus pneumoniae colonization prevalence among HIV-infected Kenyan parents in the year before pneumococcal conjugate vaccine introduction.

BACKGROUND: Streptococcus pneumoniae is a leading cause of pneumonia, meningitis and sepsis in developing countries, particularly among children and HIV-infected persons. Pneumococcal oropharyngeal (OP) or nasopharyngeal (NP) colonization is a precursor to development of invasive disease. New conjugate vaccines hold promise for reducing colonization and disease. METHODS: Prior to introduction of 10-valent pneumococcal conjugate vaccine (PCV10), we conducted a cross-sectional survey among HIV-infected parents of children <5 years old in rural Kenya. Other parents living with an HIV-infected adult were also enrolled. After broth enrichment, NP and OP swabs were cultured for pneumococcus. Serotypes were identified by Quellung. Antimicrobial susceptibility was performed using broth microdilution. RESULTS: We enrolled 973 parents; 549 (56.4%) were HIV-infected, 153 (15.7%) were HIV-uninfected and 271 (27.9%) had unknown HIV status. Among HIV-infected parents, the median age was 32 years (range 15-74) and 374/549 (68%) were mothers. Pneumococci were isolated from 237/549 (43.2%) HIV-infected parents and 41/153 (26.8%) HIV-non-infected parents (p = 0.0003). Colonization with PCV10 serotypes was not significantly more frequent in HIV-infected (12.9%) than HIV-uninfected parents (11.8%; p = 0.70). Among HIV-infected parents, cooking site separate from sleeping area and CD4 count >250 were protective (OR = 0.6; 95% CI 0.4, 0.9 and OR = 0.5; 95% CI 0.2, 0.9, respectively); other associations were not identified. Among 309 isolates tested from all parents, 255 (80.4%) were penicillin non-susceptible (MIC ≥0.12 µg/ml). CONCLUSIONS: Prevalence of pneumococcal colonization is high among HIV-infected parents in rural Kenya. If young children are the pneumococcal reservoir for this population, PCV10 introduction may reduce vaccine-type colonization and disease among HIV-infected parents through indirect protection.

Plasmodium falciparum Protein Microarray Antibody Profiles Correlate With Protection From Symptomatic Malaria in Kenya.

BACKGROUND: Immunoglobulin G antibodies (Abs) to Plasmodium falciparum antigens have been associated with naturally acquired immunity to symptomatic malaria. METHODS: We probed protein microarrays covering 824 unique P. falciparum protein features with plasma from residents of a community in Kenya monitored for 12 weeks for (re)infection and symptomatic malaria after administration of antimalarial drugs. P. falciparum proteins recognized by Abs from 88 children (aged 1-14 years) and 86 adults (aged ≥ 18 years), measured at the beginning of the observation period, were ranked by Ab signal intensity. RESULTS: Abs from immune adults reacted with a total 163 of 824 P. falciparum proteins. Children gradually acquired Abs to the full repertoire of antigens recognized by adults. Abs to some antigens showed high seroconversion rates, reaching maximal levels early in childhood, whereas others did not reach adult levels until adolescence. No correlation between Ab signal intensity and time to (re)infection was observed. In contrast, Ab levels to 106 antigens were significantly higher in children who were protected from symptomatic malaria compared with those who were not. Abs to antigens predictive of protection included P. falciparum erythrocyte membrane protein 1, merozoite surface protein (MSP) 10, MSP2, liver-stage antigen 3, PF70, MSP7, and Plasmodium helical interspersed subtelomeric domain protein. CONCLUSIONS: Protein microarrays may be useful in the search for malaria antigens associated with protective immunity.

Breast Milk as a Potential Source of Epstein-Barr Virus Transmission Among Infants Living in a Malaria-Endemic Region of Kenya.

BACKGROUND: We previously reported that infants in Kenya were infected with Epstein-Barr virus (EBV) at <6 months of age, suggesting that mothers were the likely source of transmissible virus to the infant. In this study, we investigated whether breast milk contained infectious EBV and the role of malaria in EBV shedding in breast milk. METHODS: Breast milk samples were obtained from Kenyan mothers at postpartum weeks 6, 10, 14, and 18 and analyzed for presence of infectious EBV. RESULTS: We found that the prevalence of EBV DNA and the mean EBV load were significantly higher at 6 weeks and decreased through postpartum week 18 (P < .0001). High EBV load in breast milk correlated with mothers who had Plasmodium falciparum malaria at delivery. To determine whether viral DNA was encapsidated, breast milk samples were treated with DNAse before DNA extraction. Sixty percent of samples were DNAse resistant, suggesting that the viral DNA in breast milk was encapsidated. Next, we exposed peripheral blood mononuclear cells to breast milk supernatant, which resulted in the generation of EBV-positive lymphoblastoid cell lines, indicating that the virus in breast milk was infectious. CONCLUSIONS: Our data suggest that breast milk contains infectious EBV and is a potential source of viral transmission to infants living in malaria-endemic regions.

Absence of putative artemisinin resistance mutations among Plasmodium falciparum in Sub-Saharan Africa: a molecular epidemiologic study.

Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasite's K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance.

Time-to-infection by Plasmodium falciparum is largely determined by random factors.

BACKGROUND: The identification of protective immune responses to P. falciparum infection is an important goal for the development of a vaccine for malaria. This requires the identification of susceptible and resistant individuals, so that their immune responses may be studied. Time-to-infection studies are one method for identifying putative susceptible individuals (infected early) versus resistant individuals (infected late). However, the timing of infection is dependent on random factors, such as whether the subject was bitten by an infected mosquito, as well as individual factors, such as their level of immunity. It is important to understand how much of the observed variation in infection is simply due to chance. METHODS: We analyse previously published data from a treatment-time-to-infection study of 201 individuals aged 0.5 to 78 years living in Western Kenya. We use a mathematical modelling approach to investigate the role of immunity versus random factors in determining time-to-infection in this cohort. We extend this analysis using a modelling approach to understand what factors might increase or decrease the utility of these studies for identifying susceptible and resistant individuals. RESULTS: We find that, under most circumstances, the observed distribution of time-to-infection is consistent with this simply being a random process. We find that age, method for detection of infection (PCR versus microscopy), and underlying force of infection are all factors in determining whether time-to-infection is a useful correlate of immunity. CONCLUSIONS: Many epidemiological studies of P. falciparum infection assume that the observed variation in infection outcomes, such as time-to-infection or presence or absence of infection, is determined by host resistance or susceptibility. However, under most circumstances, this distribution appears largely due to the random timing of infection, particularly in children. More direct measurements, such as parasite growth rate, may be more useful than time-to-infection in segregating patients based on their level of immunity.

Decreased growth rate of P. falciparum blood stage parasitemia with age in a holoendemic population.

In malaria holoendemic settings, decreased parasitemia and clinical disease is associated with age and cumulative exposure. The relative contribution of acquired immunity against various stages of the parasite life cycle is not well understood. In particular, it is not known whether changes in infection dynamics can be best explained by decreasing rates of infection, or by decreased growth rates of parasites in blood. Here, we analyze the dynamics of Plasmodium falciparum infection after treatment in a cohort of 197 healthy study participants of different ages. We use both polymerase chain reaction (PCR) and microscopy detection of parasitemia in order to understand parasite growth rates and infection rates over time. The more sensitive PCR assay detects parasites earlier than microscopy, and demonstrates a higher overall prevalence of infection than microscopy alone. The delay between PCR and microscopy detection is significantly longer in adults compared with children, consistent with slower parasite growth with age. We estimated the parasite multiplication rate from delay to PCR and microscopy detections of parasitemia. We find that both the delay between PCR and microscopy infection as well as the differing reinfection dynamics in different age groups are best explained by a slowing of parasite growth with age.

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.

Holoendemic malaria exposure is associated with altered Epstein-Barr virus-specific CD8(+) T-cell differentiation.

Coinfection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) is a major risk factor for endemic Burkitt lymphoma (eBL), still one of the most prevalent pediatric cancers in equatorial Africa. Although malaria infection has been associated with immunosuppression, the precise mechanisms that contribute to EBV-associated lymphomagenesis remain unclear. In this study, we used polychromatic flow cytometry to characterize CD8(+) T-cell subsets specific for EBV-derived lytic (BMFL1 and BRLF1) and latent (LMP1, LMP2, and EBNA3C) antigens in individuals with divergent malaria exposure. No malaria-associated differences in EBV-specific CD8(+) T-cell frequencies were observed. However, based on a multidimensional analysis of CD45RO, CD27, CCR7, CD127, CD57, and PD-1 expression, we found that individuals living in regions with intense and perennial (holoendemic) malaria transmission harbored more differentiated EBV-specific CD8(+) T-cell populations that contained fewer central memory cells than individuals living in regions with little or no (hypoendemic) malaria. This profile shift was most marked for EBV-specific CD8(+) T-cell populations that targeted latent antigens. Importantly, malaria exposure did not skew the phenotypic properties of either cytomegalovirus (CMV)-specific CD8(+) T cells or the global CD8(+) memory T-cell pool. These observations define a malaria-associated aberration localized to the EBV-specific CD8(+) T-cell compartment that illuminates the etiology of eBL.

Serological markers for monitoring historical changes in malaria transmission intensity in a highly endemic region of Western Kenya, 1994-2009.

BACKGROUND: Monitoring local malaria transmission intensity is essential for planning evidence-based control strategies and evaluating their impact over time. Anti-malarial antibodies provide information on cumulative exposure and have proven useful, in areas where transmission has dropped to low sustained levels, for retrospectively reconstructing the timing and magnitude of transmission reduction. It is unclear whether serological markers are also informative in high transmission settings, where interventions may reduce transmission, but to a level where considerable exposure continues. METHODS: This study was conducted through ongoing KEMRI and CDC collaboration. Asembo, in Western Kenya, is an area where intense malaria transmission was drastically reduced during a 1997-1999 community-randomized, controlled insecticide-treated net (ITN) trial. Two approaches were taken to reconstruct malaria transmission history during the period from 1994 to 2009. First, point measurements were calculated for seroprevalence, mean antibody titre, and seroconversion rate (SCR) against three Plasmodium falciparum antigens (AMA-1, MSP-119, and CSP) at five time points for comparison against traditional malaria indices (parasite prevalence and entomological inoculation rate). Second, within individual post-ITN years, age-stratified seroprevalence data were analysed retrospectively for an abrupt drop in SCR by fitting alternative reversible catalytic conversion models that allowed for change in SCR. RESULTS: Generally, point measurements of seroprevalence, antibody titres and SCR produced consistent patterns indicating that a gradual but substantial drop in malaria transmission (46-70%) occurred from 1994 to 2007, followed by a marginal increase beginning in 2008 or 2009. In particular, proportionate changes in seroprevalence and SCR point estimates (relative to 1994 baseline values) for AMA-1 and CSP, but not MSP-119, correlated closely with trends in parasite prevalence throughout the entire 15-year study period. However, retrospective analyses using datasets from 2007, 2008 and 2009 failed to detect any abrupt drop in transmission coinciding with the timing of the 1997-1999 ITN trial. CONCLUSIONS: In this highly endemic area, serological markers were useful for generating accurate point estimates of malaria transmission intensity, but not for retrospective analysis of historical changes. Further investigation, including exploration of different malaria antigens and/or alternative models of population seroconversion, may yield serological tools that are more informative in high transmission settings.

Modeling larval malaria vector habitat locations using landscape features and cumulative precipitation measures.

BACKGROUND: Predictive models of malaria vector larval habitat locations may provide a basis for understanding the spatial determinants of malaria transmission. METHODS: We used four landscape variables (topographic wetness index [TWI], soil type, land use-land cover, and distance to stream) and accumulated precipitation to model larval habitat locations in a region of western Kenya through two methods: logistic regression and random forest. Additionally, we used two separate data sets to account for variation in habitat locations across space and over time. RESULTS: Larval habitats were more likely to be present in locations with a lower slope to contributing area ratio (i.e. TWI), closer to streams, with agricultural land use relative to nonagricultural land use, and in friable clay/sandy clay loam soil and firm, silty clay/clay soil relative to friable clay soil. The probability of larval habitat presence increased with increasing accumulated precipitation. The random forest models were more accurate than the logistic regression models, especially when accumulated precipitation was included to account for seasonal differences in precipitation. The most accurate models for the two data sets had area under the curve (AUC) values of 0.864 and 0.871, respectively. TWI, distance to the nearest stream, and precipitation had the greatest mean decrease in Gini impurity criteria in these models. CONCLUSIONS: This study demonstrates the usefulness of random forest models for larval malaria vector habitat modeling. TWI and distance to the nearest stream were the two most important landscape variables in these models. Including accumulated precipitation in our models improved the accuracy of larval habitat location predictions by accounting for seasonal variation in the precipitation. Finally, the sampling strategy employed here for model parameterization could serve as a framework for creating predictive larval habitat models to assist in larval control efforts.

Humoral and cellular immunity to Plasmodium falciparum merozoite surface protein 1 and protection from infection with blood-stage parasites.

BACKGROUND: Acquired immunity to malaria develops with increasing age and repeated infections. Understanding immune correlates of protection from malaria would facilitate vaccine development and identification of biomarkers that reflect changes in susceptibility resulting from ongoing malaria control efforts. METHODS: The relationship between immunoglobulin G (IgG) antibody and both interferon γ (IFN-γ) and interleukin 10 (IL-10) responses to the 42-kD C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP142) and the risk of (re)infection were examined following drug-mediated clearance of parasitemia in 94 adults and 95 children in an area of holoendemicity of western Kenya. RESULTS: Positive IFN-γ enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot assay (ELISPOT) responses to MSP142 3D7 were associated with delayed time to (re)infection, whereas high-titer IgG antibodies to MSP142 3D7 or FVO alleles were not independently predictive of the risk of (re)infection. When IFN-γ and IL-10 responses were both present, the protective effect of IFN-γ was abrogated. A Cox proportional hazard model including IFN-γ, IL-10, MSP142 3D7 IgG antibody responses, hemoglobin S genotype, age, and infection status at baseline showed that the time to blood-stage infection correlated positively with IFN-γ responses and negatively with IL-10 responses, younger age, and asymptomatic parasitemia. CONCLUSIONS: Evaluating combined allele-specific cellular and humoral immunity elicited by malaria provides a more informative measure of protection relative to evaluation of either measure alone.

Interleukin (IL)-13 promoter polymorphisms (-7402 T/G and -4729G/A) condition susceptibility to pediatric severe malarial anemia but not circulating IL-13 levels.

In holoendemic Plasmodium falciparum transmission areas such as western Kenya, severe malarial anemia [SMA, hemoglobin (Hb) < 6.0 g/dL, with any density parasitemia] is the most common clinical manifestation of severe malaria resulting in high rates of pediatric morbidity and mortality in these regions. Previous studies associated interleukin (IL)-13 with pathogenesis of different infectious diseases, including P. falciparum malaria. However, the functional roles of polymorphic variants within the IL-13 promoter in conditioning susceptibility to SMA remain largely unexplored. As such, the association between the IL-13 variants -7402 T/G (rs7719175) and -4729G/A (rs3091307) and susceptibility to SMA was determined in children (n = 387) presenting with clinical symptoms of falciparum malaria and resident in a holoendemic transmission region in western Kenya. Our results indicated no difference in the proportions of individual genotypes among children presenting with non-SMA (n = 222) versus SMA (n = 165). Similarly, there was no associations between the individual genotypes (-7402 T/G and -4729G/A) and SMA. Additional analyses, however, revealed that proportions of individuals with -7402 T/-4729A (TA) haplotype was significantly higher in children presenting with SMA than non-SMA group (P = 0.043). A further multivariate logistic regression analyses, controlling for confounding factors, demonstrated that carriage of the TA haplotype was associated with increased susceptibility to SMA (OR; 1.564, 95% CI; 1.023-2.389, P = 0.039). In addition, circulating levels of IL-13 were comparable between the clinical groups as well as across genotypes and haplotypes. Collectively, findings presented here suggest that haplotypes within the IL-13 promoter at -7402 T/G and -4729G/A may modulate SMA pathogenesis, but do not affect circulating IL-13 levels.