Hematocrit-Independent Sampling Enables White Blood Cell Counts from Patterned Dried Blood Spot Cards.

The accurate and efficient measurement of white blood cell (WBC) counts is vital for monitoring general patient health and can aid in the diagnosis of a range of possible infections or diseases. Even with their importance universally acknowledged, access to WBC counts is largely limited to those with access to phlebotomists and centralized clinical laboratories, which house the instruments that perform the tests. As a result, large populations of people (e.g., those that are home-bound or live in remote locations) lack facile access to testing. Dried blood spot (DBS) cards are often used to bridge these gaps in access to testing by offering the ability to collect blood at home for ambient shipping to laboratories. However, it is well understood that these cards, which are prepared from cellulose cardstocks without further modification, suffer from variabilities in accuracy and precision due to uncontrolled sample spreading and hematocrit effects, which have hindered their use to determine WBC counts. In this paper, we present a method to obtain an accurate WBC count using a patterned dried blood spot (pDBS) card, which comprises collection zones that meter volumes of dried blood. Using an input volume of 75 µL of whole blood, we demonstrate that, unlike the gold standard DBS card (Whatman 903), our pDBS design allows for the collection of replicate zones containing a reproducible, average volume of dried blood (12.1 µL, 7.8% CV) over the range of hematocrits from 25 to 55%. We then used qPCR to quantify the 18S rRNA gene to determine WBC counts from the volumes of blood that are metered in pDBS zones. We observe that WBC counts generated from our method are comparable to those measured with a HemoCue point-of-care WBC analyzer. Our approach to using pDBS cards as a blood collection device has the potential to support at-home sampling and other patient populations that need WBC counts but lack access to clinical facilities.

A deep sequencing approach to estimate Plasmodium falciparum complexity of infection (COI) and explore apical membrane antigen 1 diversity.

BACKGROUND: Humans living in regions with high falciparum malaria transmission intensity harbour multi-strain infections comprised of several genetically distinct malaria haplotypes. The number of distinct malaria parasite haplotypes identified from an infected human host at a given time is referred to as the complexity of infection (COI). In this study, an amplicon-based deep sequencing method targeting the Plasmodium falciparum apical membrane antigen 1 (pfama1) was utilized to (1) investigate the relationship between P. falciparum prevalence and COI, (2) to explore the population genetic structure of P. falciparum parasites from malaria asymptomatic individuals participating in the 2007 Demographic and Health Survey (DHS) in the Democratic Republic of Congo (DRC), and (3) to explore selection pressures on geospatially divergent parasite populations by comparing AMA1 amino acid frequencies in the DRC and Mali. RESULTS: A total of 900 P. falciparum infections across 11 DRC provinces were examined. Deep sequencing of both individuals, for COI analysis, and pools of individuals, to examine population structure, identified 77 unique pfama1 haplotypes. The majority of individual infections (64.5%) contained polyclonal (COI > 1) malaria infections based on the presence of genetically distinct pfama1 haplotypes. A minimal correlation between COI and malaria prevalence as determined by sensitive real-time PCR was identified. Population genetic analyses revealed extensive haplotype diversity, the vast majority of which was shared across the sites. AMA1 amino acid frequencies were similar between parasite populations in the DRC and Mali. CONCLUSIONS: Amplicon-based deep sequencing is a useful tool for the detection of multi-strain infections that can aid in the understanding of antigen heterogeneity of potential malaria vaccine candidates, population genetics of malaria parasites, and factors that influence complex, polyclonal malaria infections. While AMA1 and other diverse markers under balancing selection may perform well for understanding COI, they may offer little geographic or temporal discrimination between parasite populations.

The hunt for protective correlates of immunity to Plasmodium falciparum malaria.

Determining an immunologic correlate of protection against Plasmodium falciparum malaria has been the holy grail of natural infection studies, and sought after as an endpoint for malaria vaccine trials. An in vitro assay that provides an accurate and precise assessment of protective immunity to malaria would make smaller, short-duration studies feasible, rather than the currently powered study designs that use morbidity or mortality as outcomes. Such a biomarker would be especially desirable in situations where malaria control measures that result in decreases in clinical endpoints and putatively waning protective immunity have been implemented. In an article published in BMC Medicine, Osier and colleagues addressed this problem, and demonstrated that antibodies promoting opsonic phagocytosis of merozoites provide a functional link between antigen-specific responses and protection. Understanding the mechanisms conferring protection against malaria not only improves our knowledge of basic human immunology, but promises to help in the design of an effective malaria vaccine.

Longitudinal and Cross-sectional Analyses of Asymptomatic HIV-1/Malaria Co-infection in Kisumu County, Kenya.

Individuals infected with HIV-1 experience more frequent and more severe episodes of malaria and are likely to harbor asymptomatic parasitemia, thus potentially making them more efficient reservoirs of malaria. Two studies (cross-sectional and longitudinal) were designed in sequence between 2015-2018 and 2018-2020, respectively, to test the hypothesis that HIV-1 infected individuals have higher prevalence of asymptomatic parasitemia and gametocytemia than the HIV-1 negatives. This article describes the overall design of the two studies, encompassing data for the longitudinal study and additional data to the previously published baseline data for the cross-sectional study. In the cross-sectional study, HIV-1 positive participants were significantly older, more likely to be male, and more likely to have parasitemia relative to HIV-1 negatives (P < 0.01). In the longitudinal study, 300 participants were followed for 6 months. Of these, 102 were HIV-1 negative, 106 were newly diagnosed HIV-1 positive, and 92 were HIV-1 positive and on antiretroviral therapy, including antifolates, at enrollment. Overall parasitemia positivity at enrollment was 17.3% (52/300). Of these, 44% (23/52) were HIV-1 negative, 52% (27/52) were newly diagnosed HIV-1 positives, and only 4% (2/52) were HIV-1 positive and on treatment. Parasitemia for those on stable antiretroviral therapy was significantly lower (hazard ratio: 0.51, P < 0.001), compared with the HIV-1-negatives. On follow-up, there was a significant decline in parasitemia prevalence (hazard ratio: 0.74, P < 0.001) among the HIV patients newly initiated on antiretroviral therapy including trimethoprim-sulfamethoxasole. These data highlight the impact of HIV-1 and HIV treatment on asymptomatic parasitemia over time.

Quality and Integrated Service Delivery: A Cross-Sectional Study of the Effects of Malaria and Antenatal Service Quality on Malaria Intervention Use in Sub-Saharan Africa.

Using regionally linked facility and household surveys, we measured the quality of integrated antenatal care and malaria in pregnancy services in Kenya, Namibia, Senegal, and Tanzania. We examined country heterogeneities for the association of integrated antenatal and malaria service quality scores with insecticide-treated bed net (ITN) use in pregnant women and children under-five and intermittent preventive treatment in pregnancy (IPTp-2) uptake. Malaria in pregnancy service quality was low overall. Our findings suggest modest, positive associations between malaria in pregnancy quality and ITN use and IPTp-2 uptake across pooled models and for most studied countries, with evidence of heterogeneity in the strength of associations and relevant confounding factors. Antenatal care quality generally was not associated with the study outcomes, although a positive interaction with malaria in pregnancy quality was present for pooled ITN use models. The improved quality of malaria services delivered during formal antenatal care can help address low coverage and usage rates of preventive malaria interventions in pregnancy and childhood. Study findings may be used to target quality improvement efforts at the sub-national level. Study methods may be adapted to identify low-performing facilities for intervention and adaption to other areas of care, such as HIV/AIDS, child immunizations, and postnatal care.

Longitudinal impact of asymptomatic malaria/HIV-1 co-infection on Plasmodium falciparum gametocyte transcript expression and transmission to Anopheles mosquitoes.

Despite significant developments towards malaria reduction, parasite transmission in the common context of HIV-1 co-infection and treatment for one or both infections has not been fully characterized. This is particularly important given that HIV-1 and malaria chemotherapies have the potential to alter gametocyte burden and mosquito infectivity. In this study, we examined 782 blood samples collected from a longitudinal cohort of 300 volunteers with asymptomatic parasitemia seeking HIV testing or treatment in the endemic region of Kisumu, Kenya, to define the impacts of HIV-1-malaria co-infection, antiretroviral therapy (ART) plus trimethoprim-sulfamethoxazole (TS) and the antimalarials artemether/lumefantrine (AL) on Plasmodium falciparum gametocyte transcript prevalence and parasite transmission to the African malaria mosquito Anopheles gambiae. Volunteers were assigned to three distinct HIV-1 groups: HIV-1 positive on treatment, HIV-1 positive newly diagnosed, and HIV-1 negative. Volunteers were monitored monthly over the course of six months. Using our highly sensitive digital droplet PCR (ddPCR) assay of three gametocyte specific transcript markers, we detected gametocyte transcripts in 51.1% of 18S positive volunteers across all study groups and time points. After correcting for multiple comparisons, the factors of HIV-1 status, time, CD4+ T-cell levels and hematocrit were not predictive of gametocyte prevalence or transmission. However, among those volunteers who were newly diagnosed with HIV-1 and malaria positive by rapid diagnostic test (RDT) at enrollment, the initiation of ART/TS and AL treatment was associated with a significant reduction in gametocyte transcript prevalence in the subsequent month when compared to HIV-1 negative volunteers treated with AL. To assess gametocyte transmissibility, volunteer blood samples were used in standard membrane feeding assays (SFMA) with laboratory-reared A. gambiae, with evidence of transmission confirmed by at least one of 25 dissected mosquitoes per sample positive for at least one midgut oocyst. HIV-1 status, CD4+ T-cell levels and hematocrit were not significantly associated with successful transmission to A. gambiae. Analysis of SMFA blood samples revealed that 50% of transmission-positive blood samples failed to test positive by Plasmodium-specific 18S ribosomal RNA quantitative PCR (qPCR) and 35% failed to test positive for any gametocyte specific transcript marker by droplet digital (ddPCR), documenting that transmission occurred in the absence of molecular parasite/gametocyte detection. Overall, these findings highlight the complexity of HIV-1 malaria co-infection and the need to further define the unpredictable role of asymptomatic parasitemia in transmission to mosquitoes.

Distribution of Triatoma dimidiata sensu lato (Reduviidae: Triatominae) and Risk Factors Associated with Household Invasion in Northern Belize, Central America.

To date, Triatoma dimidiata sensu lato [Reduviidae: Triatominae (Latreille 1811)] remains the sole vector species associated with Chagas disease transmission reported from Belize. Human infection data are limited for Belize and the disease transmission dynamics have not been thoroughly investigated, yet the likelihood of autochthonous transmission is supported by the widespread collection of infected vectors from within local households. Here, we report updated infection rates of the vector population and infestation rates for villages in north and central Belize. Overall, 275 households were enrolled in an ongoing vector surveillance program. Of the 41 insects collected, 25 were PCR positive for T. cruzi, indicating an infection rate as high as 60%. To further characterize the epidemiological risk of human-vector contact, determinants of household invasion were modeled. Local households were surveyed and characterized with respect to over 25 key factors that may be associated with household infestation by T. dimidiata s.l. While final models were not strongly predictive with respect to the risk factors that were surveyed, likely due to the low number of collection observations, the presence of domestic/peri-domestic dogs, nearby light sources, and household structure materials could be the focus of continued risk assessments. In northern Belize, this vector survey lends support to T. dimidiata s.l. inhabiting sylvatic settings as opposed to the classical paradigm of domiciliated vector populations. This designation has strong implications for the local level of human exposure risk which can help guide vector surveillance and control resources.

Initiation of anti-retroviral/Trimethoprim-Sulfamethoxazole therapy in a longitudinal cohort of HIV-1 positive individuals in Western Kenya rapidly decreases asymptomatic malarial parasitemia.

Interactions between malaria and HIV-1 have important public health implications. Our previous cross-sectional studies showed significant associations between HIV-1 positivity and malarial parasitemia with an increased risk of gametocytemia. In this follow-up longitudinal study, we evaluated these associations to determine the magnitude of asymptomatic parasitemia over time, and to examine the effects of initiating Antiretroviral Therapy (ART) together with the broad-spectrum antibiotic Trimethoprim Sulfamethoxazole (TS) on asymptomatic parasitemia. 300 adult volunteers in a malaria holoendemic region in Western Kenya were enrolled and followed for six months. The study groups were composed of 102 HIV-1 negatives, 106 newly diagnosed HIV-1 positives and 92 HIV-1 positives who were already stable on ART/TS. Blood samples were collected monthly and asymptomatic malarial parasitemia determined using sensitive 18S qPCR. Results showed significantly higher malaria prevalence in the HIV-1 negative group (61.4%) (p=0.0001) compared to HIV-1 positives newly diagnosed (36.5%) and those stable on treatment (31.45%). Further, treatment with ART/TS had an impact on incidence of asymptomatic parasitemia. In volunteers who were malaria PCR-negative at enrollment, the median time to detectable asymptomatic infection was shorter for HIV-1 negatives (149 days) compared to the HIV-1 positives on treatment (171 days) (p=0.00136). Initiation of HIV treatment among the newly diagnosed led to a reduction in malarial parasitemia (expressed as 18S copy numbers/µl) by over 85.8% within one week of treatment and a further reduction by 96% after 2 weeks. We observed that while the impact of ART/TS on parasitemia was long term, treatment with antimalarial Artemether/Lumefantrine (AL) among the malaria RDT positives had a transient effect with individuals getting re-infected after short periods. As was expected, HIV-1 negative individuals had normal CD4+ levels throughout the study. However, CD4+ levels among HIV-1 positives who started treatment were low at enrollment but increased significantly within the first month of treatment. From our association analysis, the decline in parasitemia among the HIV-1 positives on treatment was attributed to TS treatment and not increased CD4+ levels per se. Overall, this study highlights important interactions between HIV-1 and malaria that may inform future use of TS among HIV-infected patients in malaria endemic regions.

Brief Report: No Differences Between Lopinavir/Ritonavir and Nonnucleoside Reverse Transcriptase Inhibitor-Based Antiretroviral Therapy on Clearance of Plasmodium falciparum Subclinical Parasitemia in Adults Living With HIV Starting Treatment (A5297).

BACKGROUND: HIV protease inhibitors anti-Plasmodium falciparum activity in adults remains uncertain. METHODS: Adults with HIV CD4+ counts >200 cells/mm3 starting antiretroviral therapy (ART) with P. falciparum subclinical parasitemia (Pf SCP) were randomized 1:1 to (step 1) protease inhibitor lopinavir/ritonavir (LPV/r)-based (arm A) or nonnucleoside reverse transcriptase inhibitor (nNRTI)-based ART (arm B) for 15 days. In step 2, participants received nNRTI-based ART and trimethoprim/sulfamethoxazole prophylaxis for 15 days. P. falciparum SCP clearance was measured by polymerase chain reaction. The Fisher exact test [95% exact confidence interval (CI)] was used to compare proportions of P. falciparum SCP clearance (<10 parasites/µL on 3 occasions within 24 hours) between LPV/r and nNRTI arms at day 15. The Kaplan-Meier method and log-rank test were used to compare time-to-clearance. RESULTS: Fifty-two adults from Kenya, Malawi, and Uganda with a median age = 31 (Q1, Q3: 24-39) years, 33% women, with baseline median CD4+ counts of 324 (259-404) cells/mm3, median HIV-1 RNA viremia of 5.18 log10 copies/mL (4.60-5.71), and median estimated P. falciparum density of 454 parasites/µL (83-2219) enrolled in the study. Forty-nine (94%) participants completed the study. At day 15, there was no statistically significant difference in the proportions of P. falciparum SCP clearance between the LPV/r (23.1% clearance; 6 of the 26) and nNRTI (26.9% clearance; 7 of the 26) arms [between-arm difference 3.9% (95% CI, -21.1% to 28.4%; P = 1.00)]. No significant difference in time-to-clearance was observed between the arms (P = 0.80). CONCLUSIONS: In a small randomized study of adults starting ART with P. falciparum SCP, no statistically significant differences were seen between LPV/r- and nNRTI-based ART in P. falciparum SCP clearance after 15 days of treatment.

HIV-1 Impact on Malaria Transmission: A Complex and Relevant Global Health Concern.

Malaria/HIV-1 co-infection has become a significant public health problem in the tropics where there is geographical overlap of the two diseases. It is well described that co-infection impacts clinical progression of both diseases; however, less is known about the impact of co-infection on disease transmission. Malaria transmission is dependent upon multiple critical factors, one of which is the presence and viability of the sexual-stage gametocyte. In this review, we summarize evidence surrounding gametocyte production in Plasmodium falciparum and the development factors and the consequential impact that HIV-1 has on malaria parasite transmission. Epidemiological and clinical evidence surrounding anemia, immune dysregulation, and chemotherapy as it pertains to co-infection and gametocyte transmission are reviewed. We discuss significant gaps in understanding that are often due to the biological complexities of both diseases as well as the lack of entomological data necessary to define transmission success. In particular, we highlight special epidemiological populations, such as co-infected asymptomatic gametocyte carriers, and the unique role these populations have in a future focused on malaria elimination and eradication.

Asymptomatic falciparum and Non-falciparum Malarial Parasitemia in Adult Volunteers with and without HIV-1 Coinfection in a Cohort Study in Western Kenya.

Asymptomatic malarial parasitemia represents the largest reservoir of infection and transmission, and the impact of coinfection with HIV-1 on this reservoir remains incompletely described. Accordingly, we sought to determine the prevalence of asymptomatic malarial parasitemia in Kombewa, Western Kenya, a region that is endemic for both malaria and HIV-1. A total of 1,762 dried blood spots were collected from asymptomatic adults in a cross-sectional study. The presence of parasitemia was first determined by a sensitive Plasmodium genus-specific 18S assay, followed by less sensitive species-specific DNA-based quantitative polymerase chain reaction (PCR) assays. The prevalence of asymptomatic malarial parasitemia by 18S genus-specific PCR assay was 64.4% (1,134/1,762). Of the 1,134 malaria positive samples, Plasmodium falciparum was the most prevalent species (57.4%), followed by Plasmodium malariae (3.8%) and Plasmodium ovale (2.6%) as single or mixed infections. As expected, the majority of infections were below the detection limit of microscopy and rapid diagnostic tests. HIV-1 prevalence was 10.6%, and we observed a significant association with malarial parasitemia by χ2 analysis (P = 0.0475). Seventy-one percent of HIV-1 infected volunteers were positive for Plasmodium 18S (132/186), with only 29% negative (54/186). In HIV-1-negative volunteers, the proportion was lower; 64% were found to be positive for 18S (998/1,569) and 36% were negative (571/1,569). Overall, the prevalence of asymptomatic malarial parasitemia in Western Kenya is high, and knowledge of these associations with HIV-1 infection are critically important for malaria elimination and eradication efforts focused on this important reservoir population.

HIV-1 Infection Is Associated With Increased Prevalence and Abundance of Plasmodium falciparum Gametocyte-Specific Transcripts in Asymptomatic Adults in Western Kenya.

As morbidity and mortality due to malaria continue to decline, the identification of individuals with a high likelihood of transmitting malaria is needed to further reduce the prevalence of malaria. In areas of holoendemic malaria transmission, asymptomatically infected adults may be infected with transmissible gametocytes. The impact of HIV-1 on gametocyte carriage is unknown, but co-infection may lead to an increase in gametocytemia. In this study, a panel of qPCR assays was used to quantify gametocyte stage-specific transcripts present in dried blood spots obtained from asymptomatic adults seeking voluntary HIV testing in Kombewa, Kenya. A total of 1,116 Plasmodium-specific 18S-positive samples were tested and 20.5% of these individuals had detectable gametocyte-specific transcripts. Individuals also infected with HIV-1 were 1.82 times more likely to be gametocyte positive (P<0.0001) and had significantly higher gametocyte copy numbers when compared to HIV-negative individuals. Additionally, HIV-1 positivity was associated with higher gametocyte prevalence in men and increased gametocyte carriage with age. Overall, these data suggest that HIV-positive individuals may have an increased risk of transmitting malaria parasites in regions with endemic malaria transmission and therefore should be at a higher priority for treatment with gametocidal antimalarial drugs.

Plasmodium falciparum DHFR and DHPS Mutations Are Associated With HIV-1 Co-Infection and a Novel DHPS Mutation I504T Is Identified in Western Kenya.

Antifolate resistance is significant in Kenya and presumed to result from extensive use and cross-resistance between antifolate antimalarials and antibiotics, including cotrimoxazole/Bactrim used for HIV-1 chemotherapy. However, little is known about antifolate-resistant malaria in the context of newly diagnosed HIV-1 co-infection prior to administration of HIV-1 chemotherapy. Blood samples from a cross-sectional study of asymptomatic adult Kenyans enrolled during voluntary HIV testing were analyzed by PCR for Plasmodium spp. More than 95% of volunteers with identifiable parasite species (132 HIV-1 co-infected) were infected with Plasmodium falciparum alone or P. falciparum with Plasmodium ovale and/or Plasmodium malariae. Deep sequencing was used to screen for mutations in P. falciparum dihydrofolate reductase (dhfr) (N51I, C59R, S108N, I164L) and dihydropteroate synthase (dhps) (S436H, A437G, K540E, A581G) from 1133 volunteers. Individual mutations in DHPS but not DHFR correlated with HIV-1 status. DHFR haplotype diversity was significantly different among volunteers by gender and HIV-1 status. DHPS haplotype diversity by HIV-1 status was significantly different between volunteers paired by age and gender, indicating that patterns of resistance were independent of these variables. Molecular simulations for a novel DHPS mutation (I504T) suggested that the mutated protein has increased affinity for the endogenous ligand DHPPP and decreased affinity for drug binding. A sub-group of monoclonal infections revealed that age and parasitemia were not correlated and enabled identification of a rare septuple-mutant haplotype (IRNL-HGEA). In our study, adult Kenyans newly diagnosed with HIV-1 infection were predominantly infected with moderately resistant P. falciparum, with patterns of infecting parasite genotypes significantly associated with HIV-1 status. Together with the discovery of DHPS I504T, these data indicate that antifolate resistance continues to evolve in Kenya. Further, they highlight the need to understand the effects of associated mutations on both fitness and resistance of P. falciparum in the context of HIV-1 co-infection to better inform treatment for asymptomatic malaria.

Using Hematology Data from Malaria Vaccine Research Trials in Humans and Rhesus Macaques (Macaca mulatta) To Guide Volume Limits for Blood Withdrawal.

Guidelines on safe volume limits for blood collection from research participants in both humans and laboratory animals vary widely between institutions. The main adverse event that may be encountered in large blood volume withdrawal is iron-deficiency anemia. Monitoring various parameters in a standard blood panel may help to prevent this outcome. To this end, we analyzed the Hgb and MCV values from 43 humans and 46 macaques in malaria vaccine research trials. Although the percentage of blood volume removed was greater for macaques than humans, macaques demonstrated an overall increase of MCV over time, indicating the ability to respond appropriately to frequent volume withdrawals. In contrast, humans showed a consistent declining trend in MCV. These declines in human MCV and Hgb were significant from the beginning to end of the study despite withdrawals that were smaller than recommended volume limits. Limiting the volume withdrawn to no more than 12.5% seemed to be sufficient for macaques, and at 14% or more individual animals tended to fail to respond appropriately to large-volume blood loss, as demonstrated by a decrease in MCV. The overall positive erythropoietic response seen in macaques was likely due to the controlled, iron-fortified diet they received. The lack of erythropoietic response in the human subjects may warrant iron supplementation or reconsideration of current blood volume withdrawal guidelines.

Dengue Virus-1 Infection Did Not Alter the Behavioral Response of Aedes aegypti (Diptera: Culicidae) to DEET.

No licensed vaccine or antiviral drug against dengue virus (DENV) is available; therefore, most of the effort to prevent this disease is focused on reducing vector-host interactions. One of the most widely accepted methods of blocking vector-human contact is to use insect repellents to interfere with mosquito host-seeking behavior. Some arboviruses can replicate in the nervous system of the vector, raising the concern that arboviral infection may alter the insect behavioral response toward chemical stimuli. Three different Aedes aegypti (L.) mosquito cohorts: DENV-1-injected, diluent-injected, and uninjected were subjected to behavioral tests using a high-throughput screening system with 2.5% DEET and 0.14% DEET on 1, 4, 7, 10, 14, and 17 d postinjection. All test cohorts exhibited significant contact irritant or escape responses when they were exposed to 2.5% or 0.14% DEET. However, we found no biologically relevant irritancy response change in DENV-1-infected Ae. aegypti mosquitoes when they were exposed to DEET. Further studies evaluating the effects of other arboviral infections on insect repellents activity are necessary in order to provide better recommendation on the prevention of vector-borne disease transmission.

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

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

Effects of Preexposure to DEET on the Downstream Blood-Feeding Behaviors of Aedes aegypti (Diptera: Culicidae) Mosquitoes.

Mosquito behavior is heavily influenced by the chemical molecules in the environment. This knowledge can be used to modify insect behaviors; particularly to reduce vector-host contact as a powerful method for disease prevention. N,N-Diethyl-meta-toluamide (DEET) is the most widely used insect repellent in the market and an excellent example of a chemical that has been used to modify insect behavior for disease prevention. However, genetic insensitivity and habituation in Aedes aegypti (L.) mosquitoes after preexposure to DEET have been reported. In this study, we investigated the effect of preexposure to DEET on the downstream blood-feeding behavior of Ae. aegypti mosquitoes and the duration of the effect. We exposed mosquitoes to four different DEET concentrations: 0.10, 0.12, 0.14, and 0.16% for 10 min then allowed the mosquitoes to blood-feed on an artificial blood-feeding system either immediately or after being held for 1, 3, 6, or 24 h following DEET exposure. We found that preexposing Ae. aegypti mosquitoes to 0.14 or 0.16% DEET lowered their blood engorgement level, but did not alter their landing and probing behavior when compared to the control test populations. The reduction in complete blood-feeding was observed at all time periods tested, but was only statistically significant at 3 and 6 h after the preexposure process. Because reduction in blood meal has been associated with increased refeeding, future studies analyzing the effect of this behavior using arbovirus-infected mosquitoes are needed to address the concern of potentially increased vectorial capacity.

Characterization of a human reference standard for antibody to Plasmodium falciparum merozoite surface protein 1(42).

Volunteers vaccinated with a candidate malaria vaccine containing merozoite surface protein 1(42) (MSP-1(42)) exhibit antibodies to MSP-1(42) that are measured by enzyme-linked immunosorbent assay (ELISA). The purpose of this study was to make a human reference standard for MSP-1(42) antibody measured in absolute quantity units using pooled plasma samples known to contain high titers of MSP-1(42) antibody based on previous ELISA results. Immobilized metal affinity chromatography was used to determine the amount of MSP-1(42) antibody in this plasma pool. Hexahistidine-tagged MSP-1(42) antigen adsorbed to nickel-chelating resin was used to capture MSP-1(42) antibody from the plasma pool. The intact MSP-1(42) antibody-antigen complexes were eluted and total IgG was measured by an ELISA standardized against purified human IgG. In this way, the human reference standard was determined to contain 48.3 microg/mL of MSP-1(42) antibody. This reference standard may be useful as a quantitative working standard for measuring MSP-1(42) antibody response in future vaccine clinical trials involving MSP-1.

Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya utilizing a novel species-specific real-time PCR assay.

BACKGROUND: Plasmodium ovale is comprised of two genetically distinct subspecies, P. ovale curtisi and P. ovale wallikeri. Although P. ovale subspecies are similar based on morphology and geographical distribution, allelic differences indicate that P. ovale curtisi and P. ovale wallikeri are genetically divergent. Additionally, potential clinical and latency duration differences between P. ovale curtisi and P. ovale wallikeri demonstrate the need for investigation into the contribution of this neglected malaria parasite to the global malaria burden. METHODS: In order to detect all P. ovale subspecies simultaneously, we developed an inclusive P. ovale-specific real-time PCR assay based on conserved regions between P. ovale curtisi and P. ovale wallikeri in the reticulocyte binding protein 2 (rbp2) gene. Additionally, we characterized the P. ovale subspecies prevalence from 22 asymptomatic malaria infections using multilocus genotyping to discriminate P. ovale curtisi and P. ovale wallikeri. RESULTS: Our P. ovale rbp2 qPCR assay validation experiments demonstrated a linear dynamic range from 6.25 rbp2 plasmid copies/microliter to 100,000 rbp2 plasmid copies/microliter and a limit of detection of 1.5 rbp2 plasmid copies/microliter. Specificity experiments showed the ability of the rbp2 qPCR assay to detect low-levels of P. ovale in the presence of additional malaria parasite species, including P. falciparum, P. vivax, and P. malariae. We identified P. ovale curtisi and P. ovale wallikeri in Western Kenya by DNA sequencing of the tryptophan-rich antigen gene, the small subunit ribosomal RNA gene, and the rbp2 gene. CONCLUSIONS: Our novel P. ovale rbp2 qPCR assay detects P. ovale curtisi and P. ovale wallikeri simultaneously and can be utilized to characterize the prevalence, distribution, and burden of P. ovale in malaria endemic regions. Using multilocus genotyping, we also provided the first description of the prevalence of P. ovale curtisi and P. ovale wallikeri in Western Kenya, a region holoendemic for malaria transmission.

Immune mechanisms in malaria: new insights in vaccine development.

Early data emerging from the first phase 3 trial of a malaria vaccine are raising hopes that a licensed vaccine will soon be available for use in endemic countries, but given the relatively low efficacy of the vaccine, this needs to be seen as a major step forward on the road to a malaria vaccine rather than as arrival at the final destination. The focus for vaccine developers now moves to the next generation of malaria vaccines, but it is not yet clear what characteristics these new vaccines should have or how they can be evaluated. Here we briefly review the epidemiological and immunological requirements for malaria vaccines and the recent history of malaria vaccine development and then put forward a manifesto for future research in this area. We argue that rational design of more effective malaria vaccines will be accelerated by a better understanding of the immune effector mechanisms involved in parasite regulation, control and elimination.