Time to Reflect on Global Health Agenda in Kenya: A Tribute to our Academic and Biomedical Research Mentors

We submit this column to present a brief biography, a tribute to three departed global health mentors who were instrumental in our careers and for the growth of biomedical research in Kenya. We briefly discuss their educational backgrounds and put forth a set of qualities, values, personal supportive experiences, and achievements that nurtured our careers as scientists. The mentors are Prof. Ayub Opiyo Ofulla, Dr. John F. Kennedy Vulule, and Dr. Peter Odada Sumba. We appeal to the community of researchers in biomedical sciences, global health, and epidemiology who study a particular disease or health risk (conducting interventional and observational research) to mentor, teach, and serve as role models for upcoming scholars. There is a need for a positive and supportive attitude to create a universal environment to nurture the next generation of researchers transcending race, color, nationality, ethnicity, culture, faith, gender identities, sexual orientation, age, ability, and background

HIV infection drives IgM and IgG3 subclass bias in Plasmodium falciparum-specific and total immunoglobulin concentration in Western Kenya

BACKGROUND: HIV infection is associated with more frequent and severe episodes of malaria and may be the result of altered malaria-specific B cell responses. However, it is poorly understood how HIV and the associated lymphopenia and immune activation affect malaria-specific antibody responses. METHODS: HIV infected and uninfected adults were recruited from Bondo subcounty hospital in Western Kenya at the time of HIV testing (antiretroviral and co-trimoxazole prophylaxis naïve). Total and Plasmodium falciparum apical membrane antigen-1 (AMA1) and glutamate rich protein-R0 (GLURP-R0) specific IgM, IgG and IgG subclass concentrations was measured in 129 and 52 of recruited HIV-infected and uninfected individuals, respectively. In addition, HIV-1 viral load (VL), CD4+ T cell count, and C-reactive protein (CRP) concentration was quantified in study participants. Antibody levels were compared based on HIV status and the associations of antibody concentration with HIV-1 VL, CD4+ count, and CRP levels was measured using Spearman correlation testing. RESULTS: Among study participants, concentrations of IgM, IgG1 and IgG3 antibodies to AMA1 and GLURP-R0 were higher in HIV infected individuals compared to uninfected individuals (all p < 0.001). The IgG3 to IgG1 ratio to both AMA1 and GLURP-R0 was also significantly higher in HIV-infected individuals (p = 0.02). In HIV-infected participants, HIV-1 VL and CRP were weakly correlated with AMA1 and GLURP-R0 specific IgM and IgG1 concentrations and total (not antigen specific) IgM, IgG, IgG1, and IgG3 concentrations (all p < 0.05), suggesting that these changes are related in part to viral load and inflammation. CONCLUSIONS: Overall, HIV infection leads to a total and malaria antigen-specific immunoglobulin production bias towards higher levels of IgM, IgG1, and IgG3, and HIV-1 viraemia and systemic inflammation are weakly correlated with these changes. Further assessments of antibody affinity and function and correlation with risk of clinical malaria, will help to better define the effects of HIV infection on clinical and biological immunity to malaria

Comparison of non-magnetic and magnetic beads multiplex assay for assessment of Plasmodium falciparum antibodies

Background New reagents have emerged allowing researchers to assess a growing number of vaccine-associated immune parameters. Multiplex immunoassay(s) are emerging as efficient high-throughput assays in malaria serology. Currently, commercial vendors market several bead reagents for cytometric bead assays (CBA) but relative performances are not well published. We have compared two types of bead-based multiplex assays to measure relative antibody levels to malarial antigens. Methods Assays for the measurement of antibodies to five Plasmodium falciparum vaccine candidates using non-magnetic and magnetic fluorescent microspheres were compared for their performances with a Bio-Plex200 instrument. Mean fluorescence intensity (MFI) was determined from individuals from western Kenya and compared to known positive and negative control plasma samples. Results P. falciparum recombinant antigens were successfully coupled to both non-magnetic and magnetic beads in multiplex assays. MFIs between the two bead types were comparable for all antigens tested. Bead recovery was superior with magnetic beads for all antigens. MFI values of stored non-magnetic coupled beads did not differ from freshly coupled beads, though they showed higher levels of bead aggregation. Discussion Magnetic and non-magnetic beads performed similarly in P. falciparum antibody assays. Magnetic beads were more expensive, but had higher bead recovery, were more convenient to use, and provided rapid and easy protocol manipulation. Magnetic beads are a suitable alternative to non-magnetic beads in malarial antibody serology

Choice and Sources of Antimalarial Drugs Used for Self-medication in Kisumu, Western Kenya

Background: The choice and sources of antimalarial drugs used for self-medication has important implication to the current malaria treatment policies in Kenya. However, data on the choice of antimalarial drugs used for self-medication and their sources remains scanty. Objectives: The objectives of this study were to determine the prevalence of self-medication, the choice and sources of antimalarial drugs used for malaria self-medication in Kisumu city, Western Kenya. Methodology: This was a cross-sectional community based study, in which semi-structured questionnaires were randomly administered to 338 participants, in five administrative wards of Kisumu city. Results: Overall, 250 (74%) of the participants reported self-medication for perceived malaria illness. Of the 250 participants, 219 (87.6%) had used an antimalarial drug(s), while 31 (12.4%) took other drugs (antipyretics and herbs), which they perceived to have antimalarial effect. Artemisinin-based combination therapies (ACT), was the drug of choice for majority 154 (70.3%) of those who had self-medicated. The other antimalarials used were sulphadoxine/sulphalene-pyrimethamine 25 (11.4%), amodiaquine 11 (5%), chloroquine 5 (2.3%), quinine 2 (0.9%), dihydroartemisinin 1 (0.5%), halofantrene 1 (0.5%) and 20 (9%) of participants had used two different antimalarials. The antimalarial drugs were sourced from private pharmacies/chemists (78.4%), general retail shops (29.2%), left over drugs at home (1.6%), or friends, relatives and neighbors (2.8%). Conclusion: Self-medication for perceived malaria is prevalent in Kisumu city. ACT is the drug of choice for self-medication. However, a substantial proportion of individuals use currently ineffective antimalarials or other drugs, for example antipyretics, with no known antimalarial efficacy. Pharmacies/chemists and general retail shops are the major sources for self-prescribed drugs. Key words: Self-medication, antimalarial drugs, choices, source

Impact of Mothers’ Schistosomiasis Status During Gestation on Children’s IgG Antibody Responses to Routine Vaccines 2 Years Later and Anti-Schistosome and Anti-Malarial Responses by Neonates in Western Kenya

The potential consequences of parasitic infections on a person’s immune responsiveness to unrelated antigens are often conjectured upon in relationship to allergic responses and autoimmune diseases. These considerations sometimes extend to whether parasitic infection of pregnant women can influence the outcomes of responses by their offspring to the immunizations administered during national Expanded Programs of Immunization. To provide additional data to these discussions, we have enrolled 99 close-to-term pregnant women in western Kenya and determined their Schistosoma mansoni and Plasmodium falciparum infection status. At 2 years of age, when the initial immunization schedule was complete, we determined their children’s IgG antibody levels to tetanus toxoid, diphtheria toxoid, and measles nucleoprotein (N-protein) antigens using a multiplex assay. We also monitored antibody responses during the children’s first 2 years of life to P. falciparum MSP119 (PfMSP119), S. mansoni Soluble Egg Antigen (SEA), Ascaris suum hemoglobin (AsHb), and Strongyloides stercoralis (SsNIE). Mothers’ infections with either P. falciparum or S. mansoni had no impact on the level of antibody responses of their offspring or the proportion of offspring that developed protective levels of antibodies to either tetanus or diphtheria antigens at 2 years of age. However, children born of S. mansoni-positive mothers and immunized for measles at 9 months of age had significantly lower levels of anti-measles N-protein antibodies when they were 2 years old (p = 0.007) and a lower proportion of these children (62.5 vs. 90.2%, OR = 0.18, 95% CI = 0.04–0.68, p = 0.011) were considered positive for measles N-protein antibodies. Decreased levels of measles antibodies may render these children more susceptible to measles infection than children whose mothers did not have schistosomiasis. None of the children demonstrated responses to AsHb or SsNIE during the study period. Anti-SEA and anti-PfMSP119 responses suggested that 6 and 70% of the children acquired schistosomes and falciparum malaria, respectively, during the first 2 years of life

Effect of transmission intensity and age on subclass antibody responses to Plasmodium falciparum pre-erythrocytic and blood-stage antigens

Cytophilic immunoglobulin (IgG) subclass responses (IgG1 and IgG3) to Plasmodium falciparum antigens have been associated with protection from malaria, yet the relative importance of transmission intensity and age in generation of subclass responses to pre-erythrocytic and blood-stage antigens have not been clearly defined. We analyzed IgG subclass responses to the pre-erythrocytic antigens CSP, LSA-1, and TRAP and the blood-stage antigens AMA-1, EBA-175, and MSP-1 in asymptomatic residents age 2 years or older in stable (n=116) and unstable (n=96) transmission areas in Western Kenya. In the area of stable malaria transmission, a high prevalence of cytophilic (IgG1 and IgG3) antibodies to each antigen was seen in all age groups. Prevalence and levels of cytophilic antibodies to pre-erythrocytic and blood-stage P. falciparum antigens increased with age in the unstable transmission area, yet IgG1 and IgG3 responses to most antigens for all ages in the unstable transmission area were less prevalent and lower in magnitude than even the youngest age group from the stable transmission area. The dominance of cytophilic responses over non-cytophilic (IgG2 and IgG4) was more pronounced in the stable transmission area, and the ratio of IgG3 over IgG1 generally increased with age. In the unstable transmission area, the ratio of cytophilic to non-cytophilic antibodies did not increase with age, and tended to be IgG3-biased for pre-erythrocytic antigens yet IgG1-biased for blood-stage antigens. The differences between areas could not be attributed to active parasitemia status, as there were minimal differences in antibody responses between those positive and negative for Plasmodium infection by microscopy in the stable transmission area. Individuals in areas of unstable transmission have low cytophilic to non-cytophilic IgG subclass ratios and low IgG3:IgG1 ratios to P. falciparum antigens. These imbalances could contribute to the persistent risk of clinical malaria in these areas and serve as population-level, age-specific biomarkers of transmission

Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens

Abstract Background Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Methods Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Results Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from &lt;0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. Conclusion With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA

Altitude, not potential larval habitat availability, explains pronounced variation in Plasmodium falciparum infection prevalence in the western Kenya highlands.

Progress in malaria control has stalled over the recent years. Knowledge on main drivers of transmission explaining small-scale variation in prevalence can inform targeted control measures. We collected finger-prick blood samples from 3061 individuals irrespective of clinical symptoms in 20 clusters in Busia in western Kenya and screened for Plasmodium falciparum parasites using qPCR and microscopy. Clusters spanned an altitude range of 207 meters (1077-1284 m). We mapped potential mosquito larval habitats and determined their number within 250 m of a household and distances to households using ArcMap. Across all clusters, P. falciparum parasites were detected in 49.8% (1524/3061) of individuals by qPCR and 19.5% (596/3061) by microscopy. Across the clusters, prevalence ranged from 26% to 70% by qPCR. Three to 34 larval habitats per cluster and 0-17 habitats within a 250m radius around households were observed. Using a generalized linear mixed effect model (GLMM), a 5% decrease in the odds of getting infected per each 10m increase in altitude was observed, while the number of larval habitats and their proximity to households were not statistically significant predictors for prevalence. Kitchen located indoors, open eaves, a lower level of education of the household head, older age, and being male were significantly associated with higher prevalence. Pronounced variation in prevalence at small scales was observed and needs to be taken into account for malaria surveillance and control. Potential larval habitat frequency had no direct impact on prevalence

Increased investment in gametocytes in asymptomatic Plasmodium falciparum infections in the wet season.

BackgroundTransmission stemming from asymptomatic infections is increasingly being recognized as a threat to malaria elimination. In many regions, malaria transmission is seasonal. It is not well understood whether Plasmodium falciparum modulates its investment in transmission to coincide with seasonal vector abundance.MethodsWe sampled 1116 asymptomatic individuals in the wet season, when vectors are abundant, and 1743 in the dry season, in two sites in western Kenya, representing different transmission intensities (Chulaimbo, moderate transmission, and Homa Bay, low transmission). Blood samples were screened for P. falciparum by qPCR, and gametocytes by pfs25 RT-qPCR.ResultsParasite prevalence by qPCR was 27.1% (Chulaimbo, dry), 48.2% (Chulaimbo, wet), 9.4% (Homabay, dry), and 7.8% (Homabay, wet). Mean parasite densities did not differ between seasons (P = 0.562). pfs25 transcripts were detected in 119/456 (26.1%) of infections. In the wet season, fewer infections harbored detectable gametocytes (22.3% vs. 33.8%, P = 0.009), but densities were 3-fold higher (wet: 3.46 transcripts/uL, dry: 1.05 transcripts/uL, P &lt; 0.001). In the dry season, 4.0% of infections carried gametocytes at moderate-to-high densities likely infective (&gt; 1 gametocyte per 2 uL blood), compared to 7.9% in the wet season. Children aged 5-15 years harbored 76.7% of infections with gametocytes at moderate-to-high densities.ConclusionsParasites increase their investment in transmission in the wet season, reflected by higher gametocyte densities. Despite increased gametocyte densities, parasite density remained similar across seasons and were often below the limit of detection of microscopy or rapid diagnostic test, thus a large proportion of infective infections would escape population screening in the wet season. Seasonal changes of gametocytemia in asymptomatic infections need to be considered when designing malaria control measures