Eva H. Clark

A positive correlation between atypical memory B cells and Plasmodium falciparum transmission intensity in cross-sectional studies in Peru and Mali.

Background Antibodies that protect against Plasmodium falciparum (Pf) malaria are only acquired after years of repeated infections. The B cell biology that underlies this observation is poorly understood. We previously reported that “atypical” memory B cells are increased in children and adults exposed to intense Pf transmission in Mali, similar to what has been observed in individuals infected with HIV. In this study we examined B cell subsets of Pf -infected adults in Peru and Mali to determine if Pf transmission intensity correlates with atypical memory B cell expansion. Methodology/Principal Findings In this cross-sectional study venous blood was collected from adults in areas of zero (U.S., n = 10), low (Peru, n = 18) and high (Mali, n = 12) Pf transmission. Adults in Peru and Mali were infected with Pf at the time of blood collection. Thawed lymphocytes were analyzed by flow cytometry to quantify B cell subsets, including atypical memory B cells, defined by the cell surface markers CD19+ CD20+ CD21− CD27− CD10−. In Peru, the mean level of atypical memory B cells, as a percent of total B cells, was higher than U.S. adults (Peru mean: 5.4% [95% CI: 3.61–7.28]; U.S. mean: 1.4% [95% CI: 0.92–1.81]; p<0.0001) but lower than Malian adults (Mali mean 13.1% [95% CI: 10.68–15.57]; p = 0.0001). In Peru, individuals self-reporting ≥1 prior malaria episodes had a higher percentage of atypical memory B cells compared to those reporting no prior episodes (≥1 prior episodes mean: 6.6% [95% CI: 4.09–9.11]; no prior episodes mean: 3.1% [95% CI: 1.52–4.73]; p = 0.028). Conclusions/Significance Compared to Pf-naive controls, atypical memory B cells were increased in Peruvian adults exposed to low Pf transmission, and further increased in Malian adults exposed to intense Pf transmission. Understanding the origin, function and antigen specificity of atypical memory B cells in the context of Pf infection could contribute to our understanding of naturally-acquired malaria immunity.

Epidemiology of and impact of insecticide spraying on Chagas disease in communities in the Bolivian Chaco.

Background Chagas disease control campaigns relying upon residual insecticide spraying have been successful in many Southern American countries. However, in some areas, rapid reinfestation and recrudescence of transmission have occurred. Methodology/Principal Findings We conducted a cross-sectional survey in the Bolivian Chaco to evaluate prevalence of and risk factors for T. cruzi infection 11 years after two rounds of blanket insecticide application. We used a cubic B-spline model to estimate change in force of infection over time based on age-specific seroprevalence data. Overall T. cruzi seroprevalence was 51.7%. The prevalence was 19.8% among children 2–15, 72.7% among those 15–30 and 97.1% among participants older than 30 years. Based on the model, the estimated annual force of infection was 4.3% over the two years before the first blanket spray in 2000 and fell to 0.4% for 2001–2002. The estimated annual force of infection for 2004–2005, the 2 year period following the second blanket spray, was 4.6%. However, the 95% bootstrap confidence intervals overlap for all of these estimates. In a multivariable model, only sleeping in a structure with cracks in the walls (aOR = 2.35; 95% CI = 1.15–4.78), age and village of residence were associated with infection. Conclusions/Significance As in other areas in the Chaco, we found an extremely high prevalence of Chagas disease. Despite evidence that blanket insecticide application in 2000 may have decreased the force of infection, active transmission is ongoing. Continued spraying vigilance, infestation surveillance, and systematic household improvements are necessary to disrupt and sustain interruption of infection transmission.

Plasmodium falciparum malaria in the Peruvian Amazon, a region of low transmission, is associated with immunologic memory.

ABSTRACT The development of clinical immunity to Plasmodium falciparum malaria is thought to require years of parasite exposure, a delay often attributed to difficulties in developing protective antibody levels. In this study, we evaluated several P. falciparum vaccine candidate antigens, including apical membrane antigen 1 (AMA-1), circumsporozoite protein (CSP), erythrocyte binding antigen 175 (EBA-175), and the 19-kDa region of merozoite surface protein 1 (MSP119). After observing a more robust antibody response to MSP119, we evaluated the magnitude and longevity of IgG responses specific to this antigen in Peruvian adults and children before, during, and after P. falciparum infection. In this low-transmission region, even one reported prior infection was sufficient to produce a positive anti-MSP119 IgG response for >5 months in the absence of reinfection. We also observed an expansion of the total plasmablast (CD19+ CD27+ CD38high) population in the majority of individuals shortly after infection and detected MSP1-specific memory B cells in a subset of individuals at various postinfection time points. This evidence supports our hypothesis that effective antimalaria humoral immunity can develop in low-transmission regions.

Antibody response dynamics to the Plasmodium falciparum conserved vaccine candidate antigen, merozoite surface protein-1 C-terminal 19kD (MSP1-19kD), in Peruvians exposed to hypoendemic malaria transmission.

BackgroundIn high-transmission areas, developing immunity to symptomatic Plasmodium falciparum infections requires 2–10 years of uninterrupted exposure. Delayed malaria-immunity has been attributed to difficult-to-develop and then short-lived antibody responses.MethodsIn a study area with <0.5 P. falciparum infections/person/year, antibody responses to the MSP1-19kD antigen were evaluated and associations with P. falciparum infections in children and adults. In months surrounding and during the malaria seasons of 2003–2004, 1,772 participants received ≥6 active visits in one study-year. Community-wide surveys were conducted at the beginning and end of each malaria season, and weekly active visits were completed for randomly-selected individuals each month. There were 79 P. falciparum infections with serum samples collected during and approximately one month before and after infection. Anti-MSP1-19kD IgG levels were measured by ELISA.ResultsThe infection prevalence during February-July was similar in children (0.02–0.12 infections/person/month) and adults (0.03–0.14 infections/person/month) and was negligible in the four-month dry season. In children and adults, the seroprevalence was maintained in the beginning (children = 28.9%, adults = 61.8%) versus ending malaria-season community survey (children = 26.7%, adults = 64.6%). Despite the four-month non-transmission season, the IgG levels in Plasmodium-negative adults were similar to P. falciparum-positive adults. Although children frequently responded upon infection, the transition from a negative/low level before infection to a high level during/after infection was slower in children. Adults and children IgG-positive before infection had reduced symptoms and parasite density.ConclusionIndividuals in low transmission areas can rapidly develop and maintain αMSP1-19kD IgG responses for >4 months, unlike responses reported in high transmission study areas. A greater immune capacity might contribute to the frequent asymptomatic P. falciparum infections in this Peruvian population.

The Plasmodium falciparum merozoite surface protein-1 19 KD antibody response in the Peruvian Amazon predominantly targets the non-allele specific, shared sites of this antigen.

BackgroundPlasmodium falciparum re-emerged in Iquitos, Peru in 1994 and is now hypoendemic (< 0.5 infections/person/year). Purportedly non-immune individuals with discrete (non-overlapping) P. falciparum infections can be followed using this population dynamic. Previous work demonstrated a strong association between this populations antibody response to Pf MSP1-19KD and protection against febrile illness and parasitaemia. Therefore, some selection for Pf MSP1-19KD allelic diversity would be expected if the protection is to allele-specific sites of Pf MSP1-19KD. Here, the potential for allele-specific polymorphisms in this population is investigated, and the allele-specificity of antibody responses to Pf MSP1-19KD are determined.MethodsThe 42KD region in Pf MSP1 was genotyped from 160 individual infections collected between 2003 and 2007. Additionally, the polymorphic block 2 region of Pfmsp1 (Pfmsp1-B2) was genotyped in 781 infection-months to provide a baseline for population-level diversity. To test whether Pf MSP1-19KD genetic diversity had any impact on antibody responses, ELISAs testing IgG antibody response were performed on individuals using all four allele-types of Pf MSP1-19KD. An antibody depletion ELISA was used to test the ability of antibodies to cross-react between allele-types.ResultsDespite increased diversity in Pfmsp1-B2, limited diversity within Pfmsp1-42KD was observed. All 160 infections genotyped were Mad20-like at the Pfmsp1-33KD locus. In the Pfmsp1-19KD locus, 159 (99.4%) were the Q-KSNG-F haplotype and 1 (0.6%) was the E-KSNG-L haplotype. Antibody responses in 105 individuals showed that Q-KNG and Q-TSR alleles generated the strongest immune responses, while Q-KNG and E-KNG responses were more concordant with each other than with those from Q-TSR and E-TSR, and vice versa. The immuno-depletion ELISAs showed all samples responded to the antigenic sites shared amongst all allelic forms of Pf MSP1-19KD.ConclusionsA non-allele specific antibody response in Pf MSP1-19KD may explain why other allelic forms have not been maintained or evolved in this population. This has important implications for the use of Pf MSP1-19KD as a vaccine candidate. It is possible that Peruvians have increased antibody responses to the shared sites of Pf MSP1-19KD, either due to exposure/parasite characteristics or due to a human-genetic predisposition. Alternatively, these allelic polymorphisms are not immune-specific even in other geographic regions, implying these polymorphisms may be less important in immune evasion that previous studies suggest.

Hyperendemic Chagas Disease and the Unmet Need for Pacemakers in the Bolivian Chaco

The Southern Cone Initiative to control Chagas disease is a major public health success story [1]. Household insecticide spray programs have greatly diminished infestation by Triatoma infestans, the major domestic vector, blood bank and congenital Chagas disease screening have been implemented widely, and the estimated prevalence of Trypanosoma cruzi infection has fallen by more than 50% in the last 20 years [2]. Morbidity and mortality from Chagas cardiomyopathy, the most serious manifestation of T. cruzi infection, have declined progressively with disruption of domestic vector-borne transmission and increasing availability of advanced cardiac care [3]. The major exception to this pattern of success is the Gran Chaco, an ecological zone shared among Argentina, Bolivia, and Paraguay, and host to the highest rates of vector infestation and T. cruzi infection ever reported. Challenges include rapid reinfestation after spray campaigns, insecticide resistance, and sylvatic Tri. infestans populations [4]–[6]. Chagas cardiomyopathy occurs in an estimated 20%–30% of T. cruzi-infected individuals and features a chronic inflammatory process affecting the conduction system and myocardium [2], [7]. The earliest signs are typically bundle branch blocks and segmental wall motion abnormalities, usually beginning in early adulthood [8]. Later, patients may develop ventricular tachycardia, severe sinus bradycardia, high degree atrioventricular block (AVB), apical aneurysm, progressive dilated cardiomyopathy, and thromboemboli [2]. Syncope from heart block or severe bradycardia is common. Once conduction system abnormalities or arrhythmias are present, patients have shortened survival; signs of left ventricular dysfunction are associated with high short-term mortality [2]. Where accessible, advanced cardiac management has significantly improved the prognosis for patients with Chagas cardiomyopathy [9]. In 2011, we conducted a study of T. cruzi infection in seven villages in the Bolivian Chaco [6]. T. cruzi infection prevalence was 26.2% among residents younger than 20, 85.4% among 20–29-year-olds and 96.7% among participants 30 years or older. We then offered electrocardiograms (ECGs) to all study participants 20 years or older with T. cruzi infection. A total of 327 seropositive participants, 59% of infected study participants, had an ECG evaluated by the study cardiologists. Within a few days, we began to see patients who urgently needed pacemakers. In this article, we present a patient case report and a brief description of other patients found to need pacemakers, and we place these findings in the larger context of the Bolivian Chaco and the impact of Chagas heart disease there.

Circulating Serum Markers and QRS Scar Score in Chagas Cardiomyopathy

Approximately 8 million people have Trypanosoma cruzi infection, and nearly 30% will manifest Chagas cardiomyopathy (CC). Identification of reliable early indicators of CC risk would enable prioritization of treatment to those with the highest probability of future disease. Serum markers and electrocardiogram (EKG) changes were measured in 68 T. cruzi-infected individuals in various stages of cardiac disease and 17 individuals without T. cruzi infection or cardiac disease. T. cruzi-infected individuals were assigned to stage A (normal EKG/chest x-ray [CXR]), B (abnormal EKG/normal CXR), or C (abnormal EKG/cardiac structural changes). Ten serum markers were measured using enzyme-linked immunosorbent assay (ELISA)/Luminex, and QRS scores were calculated. Higher concentrations of transforming growth factor-β1 (TGFβ1), and TGFβ2 were associated with stage B compared with stage A. Matrix Metalloproteinase 2 (MMP2), Tissue Inhibitors of MMP 1, QRS score, and Brain Natriuretic Protein rose progressively with increasing CC severity. Elevated levels of several markers of cardiac damage and inflammation are seen in early CC and warrant additional evaluation in longitudinal studies.

Global Health Research in Narrative: A Qualitative Look at the FICRS-F Experience

For American professional and graduate health sciences trainees, a mentored fellowship in a low- or middle-income country (LMIC) can be a transformative experience of personal growth and scientific discovery. We invited 86 American trainees in the Fogarty International Clinical Research Scholars and Fellows Program and Fulbright-Fogarty Fellowship 2011-2012 cohorts to contribute personal essays about formative experiences from their fellowships. Nine trainees contributed essays that were analyzed using an inductive approach. The most frequently addressed themes were the strong continuity of research and infrastructure at Fogarty fellowship sites, the time-limited nature of this international fellowship experience, and the ways in which this fellowship period was important for shaping future career planning. Trainees also addressed interaction with host communities vis-à-vis engagement in project implementation. These qualitative essays have contributed insights on how a 1-year mentored LMIC-based research training experience can influence professional development, complementing conventional evaluations. Full text of the essays is available at http://fogartyscholars.org/.

Field Evaluation of the InBios Chagas Detect Plus Rapid Test in Serum and Whole-Blood Specimens in Bolivia

ABSTRACT Trypanosoma cruzi causes Chagas disease, which affects an estimated 7 million to 8 million people. Chagas disease is endemic throughout Latin America, with the highest prevalence in Bolivia. Conventional diagnosis requires a well-equipped laboratory with experienced personnel. We evaluated the Chagas Detect Plus (CDP) (InBios, Seattle, WA), a rapid immunochromatographic assay for IgG antibodies to T. cruzi. CDP performance was compared to infection status based on results obtained by indirect hemagglutination assay, immunofluorescent-antibody test, and enzyme-linked immunosorbent assay. Confirmed infection required positive results by at least 2 conventional assays. We used specimens from adults of both sexes in a general hospital in the city of Santa Cruz and from pregnant women in a hospital and children in villages in the Bolivian Chaco, an area of hyperendemicity. CDP was performed in paired whole-blood and serum specimens from 385 individuals in the two hospital studies and in 200 serum specimens from the community study. CDP showed sensitivities/specificities of 96.2% (95% confidence interval, 92.7 to 98.4)/98.8% (95.9 to 99.9) in whole blood and 99.3% (97.5 to 99.9)/96.9% (94.2 to 98.6) in serum, with no differences by sex, age group, or study site. CDP showed excellent sensitivity and specificity in our study population, comparable to those of conventional serology. The test is reliable for field surveys, requires no laboratory equipment, and performed well in serum and whole blood. The CDP could also be used for accurate maternal screening to identify neonates at risk of congenital transmission. CDP performance data in diverse geographic areas are needed to strengthen the evidence base for its use.

Toxicological, Enzymatic, and Molecular Assessment of the Insecticide Susceptibility Profile of Triatoma infestans (Hemiptera: Reduviidae, Triatominae) Populations From Rural Communities of Santa Cruz, Bolivia

Abstract A wide range of insecticide resistance profiles has been reported across Bolivian domestic and sylvatic populations of Triatoma infestans (Klug, 1834) (Hemiptera, Reduviidae), including some with levels proven to be a threat for vector control. In this work, the insecticide profile of domestic T. infestans was studied with standardized toxicological bioassays, in an area that has not undergone consistent vector control. F1 first-instar nymphs hatched in laboratory from bugs captured in three communities from the Santa Cruz Department were evaluated with different insecticides. Moreover, the enzymatic activity of esterases and cytochrome P450 monooxygenases was measured in individual insects to evaluate the possible mechanism of metabolic resistance to pyrethroids. In addition, the DNA sequence of sodium channel gene (kdr) was screened for two point mutations associated with pyrethroid resistance previously reported in T. infestans. All populations showed reduced susceptibility to deltamethrin and a-cypermethrin, albeit the RR50 values varied significantly among them. Increased P450 monooxygenases and permethrate esterases suggest the contribution, as detoxifying mechanisms, to the observed resistance to deltamethrin in all studied populations. No individuals presented either mutation associated to resistance in the kdr gene. The level of susceptibility to a-cypermethrin, the insecticide used by the local vector control program, falls within an acceptable range to continue its use in these populations. However, the observed RR50 values evidence the possibility of selection for resistance to pyrethroids, especially to deltamethrin. Consequently, the use of pyrethroid insecticides should be closely monitored in these communities, which should be kept under entomological surveillance and sustained interventions.