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.

Maternal infection causes dysfunctional BCR signaling in male offspring due to aberrant Xist expression.

Infections during pregnancy with pathogens such as helminths correlate with altered immune responses to common childhood immunizations. However, the molecular mechanisms that underlie this remain unknown. Using our murine model of maternal schistosomiasis, when immunized, males from infected mothers had a lower frequency of antigen-specific germinal center B cells and downregulation of transcripts downstream of BCR signaling compared to males from uninfected mothers. This is driven by a reduction in developing B cell populations within the bone marrow of pups from infected mothers. Males from infected mothers were impacted to a greater extent than their female littermate counterparts. We found this defect to be caused by aberrant expression of the long non-coding RNA Xist in males leading to dysregulated Igα expression on developing B cells. This, for the first time, links dysfunctional BCR signaling with Xist expression, while also proposing a detrimental function for Xist expression in males.

Impact of parasite genomic dynamics on the sensitivity of Plasmodium falciparum isolates to piperaquine and other antimalarial drugs.

BACKGROUND: Dihydroartemisinin-piperaquine (DHA-PPQ) is an alternative first-line antimalarial to artemether-lumefantrine in Kenya. However, recent reports on the emergence of PPQ resistance in Southeast Asia threaten its continued use in Kenya and Africa. In line with the policy on continued deployment of DHA-PPQ, it is imperative to monitor the susceptibility of Kenyan parasites to PPQ and other antimalarials. METHODS: Parasite isolates collected between 2008 and 2021 from individuals with naturally acquired P. falciparum infections presenting with uncomplicated malaria were tested for in vitro susceptibility to piperaquine, dihydroartemisinin, lumefantrine, artemether, and chloroquine using the malaria SYBR Green I method. A subset of the 2019-2021 samples was further tested for ex vivo susceptibility to PPQ using piperaquine survival assay (PSA). Each isolate was also characterized for mutations associated with antimalarial resistance in Pfcrt, Pfmdr1, Pfpm2/3, Pfdhfr, and Pfdhps genes using real-time PCR and Agena MassARRAY platform. Associations between phenotype and genotype were also determined. RESULTS: The PPQ median IC50 interquartile range (IQR) remained stable during the study period, 32.70 nM (IQR 20.2-45.6) in 2008 and 27.30 nM (IQR 6.9-52.8) in 2021 (P=0.1615). The median ex vivo piperaquine survival rate (IQR) was 0% (0-5.27) at 95% CI. Five isolates had a PSA survival rate of ≥10%, consistent with the range of PPQ-resistant parasites, though they lacked polymorphisms in Pfmdr1 and Plasmepsin genes. Lumefantrine and artemether median IC50s rose significantly to 62.40 nM (IQR 26.9-100.8) (P = 0.0201); 7.00 nM (IQR 2.4-13.4) (P = 0.0021) in 2021 from 26.30 nM (IQR 5.1-64.3); and 2.70 nM (IQR 1.3-10.4) in 2008, respectively. Conversely, chloroquine median IC50s decreased significantly to 10.30 nM (IQR 7.2-20.9) in 2021 from 15.30 nM (IQR 7.6-30.4) in 2008, coinciding with a decline in the prevalence of Pfcrt 76T allele over time (P = 0.0357). The proportions of piperaquine-resistant markers including Pfpm2/3 and Pfmdr1 did not vary significantly. A significant association was observed between PPQ IC50 and Pfcrt K76T allele (P=0.0026). CONCLUSIONS: Circulating Kenyan parasites have remained sensitive to PPQ and other antimalarials, though the response to artemether (ART) and lumefantrine (LM) is declining. This study forms a baseline for continued surveillance of current antimalarials for timely detection of resistance.

Why Climate Action Is Global Health Action.

The impacts of climate change on global health and populations are far-reaching, yet they disproportionately affect vulnerable groups, thereby exacerbating disparities. As humanity reckons with the emergency of climate change, our global health community needs to contend with our own contributions to greenhouse gas emissions. We know that transformation is possible and that climate action is the antidote to the existential challenge. As a global health community, we have an immense opportunity, responsibility, and commitment to lead, support, inspire, and empower climate action, research, and innovation that align deeply with our mission and core values.

Expression of CD117 (c-Kit) on Circulating B Cells in Pediatric Schistosomiasis.

Few B cells express CD27, the primary marker for memory B cells, in pediatric schistosomiasis, suggesting B cell malfunction. This study further demonstrates unexpected high expression of CD117 on circulating B cells in children highly exposed to Schistosoma mansoni infectious larvae. CD117 is expressed by immature or lymphoma B cells, but not by mature, circulating cells. We therefore sought to define the significance of CD117 on blood B cells. We found that CD117-positive (CD117+) B cells increased with the intensity of schistosome infection. In addition, CD117 expression was reduced on CD23+ B cells previously shown to correlate with resistance to infection. Stimulation with a panel of cytokines demonstrated that CD117 levels were upregulated in response to a combination of interleukin 4 (IL-4) and stem cell factor (SCF), the ligand for CD117, whereas IL-2 led to a reduction. In addition, stimulation with SCF generally reduced B cell activation levels. Upon further investigation, it was established that multiple circulating cells expressed increased levels of CD117, including monocytes, neutrophils, and eosinophils, and expression levels correlated with that of B cells. Finally, we identified a population of large circulating cells with features of reticulocytes. Overall, our results suggest that hyperexposure to intravascular parasitic worms elicits immature cells from the bone marrow. Levels of SCF were shown to reduce as children began to transition through puberty. The study results pose an explanation for the inability of children to develop significant immunity to infection until after puberty.

Potential Utility of Systemic Plasma Biomarkers for Evaluation of Pediatric Schistosomiasis in Western Kenya.

Introduction: Current diagnostic tools for schistosomiasis are limited, and new tests are necessary to enhance disease diagnosis and surveillance. Identification of novel disease-specific biomarkers may facilitate the development of such tests. We evaluated a panel of biomarkers used in sepsis and parasitic diseases for their potential suitability in the diagnosis of schistosomiasis. Objective: The study evaluated the levels of systemic plasma biomarkers in relation to Schistosoma mansoni infection and parasite burden. Methods: Six biomarkers were measured in the plasma of children from schistosomiasis-endemic regions using ELISA. The concentration of soluble CD23 (sCD23) and lipopolysaccharide (LPS) was tested in 199 and 124 plasma samples, respectively, while interleukin-6 (IL-6), soluble triggering receptor expressed on myeloid (sTREM) cells, eotaxin-1, and fatty acid-binding protein (FABP) concentrations were tested in 30 plasma samples. Results: The concentration of IL-6, eotaxin-1, FABP, and LPS was similar between schistosome-infected and uninfected children. The schistosome-infected children had higher median levels of sTREM and sCD23 as compared to uninfected children, 119.0 (29.9-208.9) versus 10.7 (0.0-73.4) (p = 0.046) and 2,549.0 (1,899.0-3,356.0) vs. 2,035.0 (1,448.0-2,939.0) (p = 0.05), respectively. In addition, sTREM was positively correlated with egg density (p = 0.017). Conclusion: Our data show that active schistosomiasis per se is associated with elevated levels of sTREM and sCD23. sTREM has potential diagnostic and prognostic values. However, these biomarkers did not distinguish between children with low egg burden and uninfected children.

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.

Physiological and proteomic profiles of Trypanosoma brucei rhodesiense parasite isolated from suramin responsive and non-responsive HAT patients in Busoga, Uganda.

Human African Trypanosomiasis (HAT) is a disease of major economic importance in Sub-Saharan Africa. The HAT is caused by Trypanosoma brucei rhodesiense (Tbr) parasite in eastern and southern Africa, with suramin as drug of choice for treatment of early stage of the disease. Suramin treatment failures has been observed among HAT patients in Tbr foci in Uganda. In this study, we assessed Tbr parasite strains isolated from HAT patients responsive (Tbr EATRO-232) and non-responsive (Tbr EATRO-734) to suramin treatment in Busoga, Uganda for 1) putative role of suramin resistance in the treatment failure 2) correlation of suramin resistance with Tbr pathogenicity and 3) proteomic pathways underpinning the potential suramin resistance phenotype in vivo. We first assessed suramin response in each isolate by infecting male Swiss white mice followed by treatment using a series of suramin doses. We then assessed relative pathogenicity of the two Tbr isolates by assessing changes pathogenicity indices (prepatent period, survival and mortality). We finally isolated proteins from mice infected by the isolates, and assessed their proteomic profiles using mass spectrometry. We established putative resistance to 2.5 mg/kg suramin in the parasite Tbr EATRO-734. We established that Tbr EATRO-734 proliferated slower and has significantly enriched pathways associated with detoxification and metabolism of energy and drugs relative to Tbr EATRO-232. The Tbr EATRO-734 also has more abundantly expressed mitochondrion proteins and enzymes than Tbr EATRO-232. The suramin treatment failure may be linked to the relatively higher resistance to suramin in Tbr EATRO-734 than Tbr EATRO-232, among other host and parasite specific factors. However, the Tbr EATRO-734 appears to be less pathogenic than Tbr EATRO-232, as evidenced by its lower rate of parasitaemia. The Tbr EATRO-734 putatively surmount suramin challenges through induction of energy metabolism pathways. These cellular and molecular processes may be involved in suramin resistance in Tbr.

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

BACKGROUND: Transmission 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. METHODS: We 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. RESULTS: Parasite 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 < 0.001). In the dry season, 4.0% of infections carried gametocytes at moderate-to-high densities likely infective (> 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. CONCLUSIONS: Parasites 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.

Antibody Correlates of Protection from Clinical Plasmodium falciparum Malaria in an Area of Low and Unstable Malaria Transmission.

Immune correlates of protection against clinical malaria are difficult to ascertain in low-transmission areas because of the limited number of malaria cases. We collected blood samples from 5,753 individuals in a Kenyan highland area, ascertained malaria incidence in this population over the next 6 years, and then compared antibody responses to 11 Plasmodium falciparum vaccine candidate antigens in individuals who did versus did not develop clinical malaria in a nested case-control study (154 cases and 462 controls). Individuals were matched by age and village. Antigens tested included circumsporozoite protein (CSP), liver-stage antigen (LSA)-1, apical membrane antigen-1 FVO and 3D7 strains, erythrocyte-binding antigen-175, erythrocyte-binding protein-2, merozoite surface protein (MSP)-1 FVO and 3D7 strains, MSP-3, and glutamate-rich protein (GLURP) N-terminal non-repetitive (R0) and C-terminal repetitive (R2) regions. After adjustment for potential confounding factors, the presence of antibodies to LSA-1, GLURP-R2, or GLURP-R0 was associated with decreased odds of developing clinical malaria (odds ratio [OR], [95% CI] 0.56 [0.36-0.89], 0.56 [0.36-0.87], and 0.77 [0.43-1.02], respectively). Levels of antibodies to LSA-1, GLURP-R2, and CSP were associated with decreased odds of developing clinical malaria (OR [95% CI]; 0.61 [0.41-0.89], 0.60 [0.43-0.84], and 0.49 [0.24-0.99], for every 10-fold increase in antibody levels, respectively). The presence of antibodies to CSP, GLURP-R0, GLURP-R2, and LSA-1 combined best-predicted protection from clinical malaria. Antibodies to CSP, GLURP-R0, GLURP-R2, and LSA-1 are associated with protection against clinical malaria in a low-transmission setting. Vaccines containing these antigens should be evaluated in low malaria transmission areas.

Antibody Profiles to P. falciparum Antigens Over Time Characterize Acute and Long-Term Malaria Exposure in an Area of Low and Unstable Transmission.

Prevalence and levels of antibodies to multiple Plasmodium falciparum antigens show promise as tools for estimating malaria exposure. In a highland area of Kenya with unstable transmission, we assessed the presence and levels of antibodies to 12 pre-erythrocytic and blood-stage P. falciparum antigens by multiplex cytometric bead assay or ELISA in 604 individuals in August 2007, with follow-up testing in this cohort in April 2008, April 2009, and May 2010. Four hundred individuals were tested at all four time points. During this period, the only substantial malaria incidence occurred from April to August 2009. Antibody prevalence in adults was high at all time points (> 70%) for apical membrane antigen 1, erythrocyte-binding antigen 175, erythrocyte-binding protein-2, glutamate rich protein (GLURP)-R2, merozoite surface protein (MSP) 1 (19), MSP-1 (42), and liver-stage antigen-1; moderate (30-70%) for GLURP-R0, MSP-3, and thrombospondin-related adhesive protein; and low (< 30%) for SE and circumsporozoite protein (CSP). Changes in community-wide malaria exposure were best reflected in decreasing antibody levels overtime for highly immunogenic antigens, and in antibody seroprevalence overtime for the less-immunogenic antigens. Over the 3 years, antibody levels to all antigens except CSP and schizont extract (SE) decreased in an age-dependent manner. Prevalence and levels of antibodies to all antigens except CSP and SE increased with age. Increases in antibody prevalence and levels to CSP and SE coincided with increases in community-wide malaria incidence. Antibody levels to multiple P. falciparum antigens decrease in the absence of consistent transmission. Multiplex assays that assess both the presence and level of antibodies to multiple pre-erythrocytic and blood-stage P. falciparum antigens may provide the most useful estimates of past and recent malaria transmission in areas of unstable transmission and could be useful tools in malaria control and elimination campaigns.

Evaluation of morbidity in Schistosoma mansoni-positive primary and secondary school children after four years of mass drug administration of praziquantel in western Kenya.

BACKGROUND: World Health Organization guidelines recommend preventive chemotherapy with praziquantel to control morbidity due to schistosomiasis. The primary aim of this cross-sectional study was to determine if 4 years of annual mass drug administration (MDA) in primary and secondary schools lowered potential markers of morbidity in infected children 1 year after the final MDA compared to infected children prior to initial MDA intervention. METHODS: Between 2012 and 2016 all students in two primary and three secondary schools within three kilometers of Lake Victoria in western Kenya received annual mass praziquantel administration. To evaluate potential changes in morbidity we measured height, weight, mid-upper arm circumference, hemoglobin levels, abdominal ultrasound, and quality of life in children in these schools. This study compared two cross-sectional samples of Schistosoma mansoni egg-positive children: one at baseline and one at year five, 1 year after the fourth annual MDA. Data were analyzed for all ages (6-18 years old) and stratified by primary (6-12 years old) and secondary (12-18 years old) school groups. RESULTS: The prevalence of multiple potential morbidity markers did not differ significantly between the egg-positive participants at baseline and those at 5 years by Mann Whitney nonparametric analysis and Fisher's exact test for continuous and categorical data, respectively. There was a small but significantly higher score in school-related quality of life assessment by year five compared to baseline by Mann Whitney analysis (P = 0.048) in 13-18 year olds where malaria-negative. However, anemia was not positively impacted by four annual rounds of MDA, but registered a significant negative outcome. CONCLUSIONS: We did not detect differences in morbidity markers measured in a population of those infected or re-infected after multiple MDA. This could have been due to their relative insensitivity or a failure of MDA to prevent morbidity among those who remain infected. High malaria transmission in this area and/or a lack of suitable methods to measure the more subtle functional morbidities caused by schistosomiasis could be a factor. Further research is needed to identify and develop well-defined, easily quantifiable S. mansoni morbidity markers for this age group.

The prevalence of intestinal parasites and associated risk factors in school-going children from informal settlements in Nakuru town, Kenya.

BACKGROUND: Intestinal parasites are a major public health problem in the developing world and have attracted increasing levels of interest from health researchers over the past decade. Epidemiology-based studies have shown that the prevalence of intestinal parasites is high and they frequently recur in regions with poor sanitation and inadequate sewerage facilities. In this study, we determined the prevalence of intestinal parasites, their egg intensities per sample, and associated risk factors in an informal settlement. METHODS: This was a cross-sectional study conducted in three randomly selected public primary schools located in the informal settlements of Nakuru town. A total of 248 stool samples were collected from asymptomatic pupils and screened, using the Kato Katz technique, for infections caused by soil-transmitted helminths (STH). A random subset of stool samples (n=96) was also screened by polymerase chain reaction (PCR) to detect intestinal protozoa. Socio-demographic variables were collected using a pre-tested structured questionnaire; these data were analysed to identify risk factors for infection. RESULTS: The overall prevalence of intestinal parasites was 17.3% (43/248 pupils). The overall prevalence of both STH and intestinal protozoan parasites was 1.2% and 41.7%, respectively. The most commonly diagnosed STH infection was Trichuris trichiura (1.2%), followed by hookworms (0.4%) and Ascaris lumbricoides (0.4%). The prevalence of intestinal protozoan parasites ranged from 0% to 38.5% and included Entamoeba histolytica, Entamoeba hartmanni, Entamoeba dispar, Giardia intestinalis, and Entamoeba coli. All infections were light, with an egg intensity <100 for each of the STH infections. The prevalence of multiple infections, including intestinal protozoan parasites, was 5.2% (n=5) and 0.4% (n=1) for STH in the subset samples. Finally, our analysis identified several significant risk factors for intestinal parasitic infections, including goat rearing (p=0.046), living in a home with an earthen floor (p=0.022), the number of rooms in the household (p=0.035), and the source of food (p=0.016). CONCLUSION: The low prevalence of intestinal parasites in the informal settlements of Nakuru may be attributed to improvements in hygiene and sanitation, deworming, and general good health practices that are facilitated by the Department of Public Health.

Performance characteristics of a modified HIV-1 drug resistance genotyping method for use in resource-limited settings.

Background: HIV-1 drug resistance (HIVDR) assays are critical components of HIV clinical management programs in the face of emerging drug resistance. However, the high costs associated with existing commercial HIVDR assays prohibit their routine usage in resource-limited settings. We present the performance characteristics of a modified commercial HIVDR testing assay. Methods: A total of 26 plasma samples were used to validate and assess the accuracy, precision, reproducibility and amplification sensitivity of a modified HIVDR assay by HIV genotyping. In addition, a cost comparison between the original and the modified assay was performed using the ingredient costing approach. Results: The performance characteristics of the modified assay were in agreement with the original assay. Accuracy, precision and reproducibility showed nucleotide sequence identity of 98.5% (confidence interval (CI), 97.9-99.1%), 98.67% (CI, 98.1-99.23) and 98.7% (CI, 98.1-99.3), respectively.  There was no difference in the type of mutations detected by the two assays (χ 2 = 2.36, p = 0.26). Precision and reproducibility showed significant mutation agreement between replicates (kappa = 0.79 and 0.78), respectively ( p < 0.05). The amplification sensitivity of the modified assay was 100% and 62.5% for viremia ≥1000 copies/ml and <1000 copies/ml respectively. Our assay modification translates to a 39.2% reduction in the cost of reagents. Conclusions: Our findings underscore the potential of modifying commercially available HIVDR testing assays into cost-effective, yet accurate assays for use in resource-limited settings.

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.

Young Adults in Endemic Areas: An Untreated Group in Need of School-Based Preventive Chemotherapy for Schistosomiasis Control and Elimination.

Parasitologic surveys of young adults in college and university settings are not commonly done, even in areas known to be endemic for schistosomiasis and soil-transmitted helminths. We have done a survey of 291 students and staff at the Kisumu National Polytechnic in Kisumu, Kenya, using the stool microscopy Kato-Katz (KK) method and the urine point-of-care circulating cathodic antigen (POC-CCA) test. Based on three stools/two KK slides each, in the 208 participants for whom three consecutive stools were obtained, Schistosoma mansoni prevalence was 17.8%. When all 291 individuals were analyzed based on the first stool, as done by the national neglected tropical disease (NTD) program, and one urine POC-CCA assay (n = 276), the prevalence was 13.7% by KK and 23.2% by POC-CCA. Based on three stools, 2.5% of 208 participants had heavy S. mansoni infections (≥400 eggs/gram feces), with heavy S. mansoni infections making up 13.5% of the S. mansoni cases. The prevalence of the soil-transmitted helminths (STH: Ascaris lumbricoides, Trichuris trichiura and hookworm) by three stools was 1.4%, 3.1%, and 4.1%, respectively, and by the first stool was 1.4%, 2.4% and 1.4%, respectively. This prevalence and intensity of infection with S. mansoni in a college setting warrants mass drug administration with praziquantel. This population of young adults is 'in school' and is both approachable and worthy of inclusion in national schistosomiasis control and elimination programs.

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.

Functional Studies of T Regulatory Lymphocytes in Human Schistosomiasis in Western Kenya.

Immunoregulation is considered a common feature of Schistosoma mansoni infections, and elevated levels of T regulatory (Treg) lymphocytes have been reported during chronic human schistosomiasis. We now report that the removal of Treg (CD4+/CD25hi/CD127low lymphocytes) from peripheral blood mononuclear cells (PBMCs) of S. mansoni-infected individuals leads to increased levels of phytohemagglutinin (PHA)-stimulated interferon gamma (IFNγ) production and decreased interleukin-10 (IL-10) responses. Exposure to schistosome antigens did not result in measurable IFNγ by either PBMC or Treg-depleted populations. Interleukin-10 responses to soluble egg antigens (SEA) by PBMC were unchanged by Treg depletion, but the depletion of Treg greatly decreased IL-10 production to soluble worm antigenic preparation (SWAP). Proliferative responses to PHA increased upon Treg removal, but responses to SEA or SWAP did not, unless only initially low responders were evaluated. Addition of anti-IL-10 increased PBMC proliferative responses to either SEA or SWAP, but did not alter responses by Treg-depleted cells. Blockade by anti-transforming growth factor-beta (TGF-ß) increased SEA but not SWAP proliferative responses by PBMC, whereas anti-TGF-ß increased both SEA- and SWAP-stimulated responses by Treg-depleted cultures. Addition of both anti-IL-10 and anti-TGF-ß to PBMC or Treg-depleted populations increased proliferation of both populations to either SEA or SWAP. These studies demonstrate that Treg appear to produce much of the antigen-stimulated IL-10, but other cell types or subsets of Treg may produce much of the TGF-ß. The elevated levels of Treg seen in chronic schistosomiasis appear functional and involve IL-10 and TGF-ß in antigen-specific immunoregulation perhaps leading to regulation of immunopathology and/or possibly decreased immunoprotective responses.

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.