Natural immunity to malaria preferentially targets the endothelial protein C receptor-binding regions of PfEMP1s.

Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an "adult-like" response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This "adult-like" response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Antenatal, intrapartum and infant azithromycin to prevent stillbirths and infant deaths: study protocol for SANTE, a 2×2 factorial randomised controlled trial in Mali.

INTRODUCTION: In high mortality settings, prophylactic azithromycin has been shown to improve birth weight and gestational age at birth when administered antenatally, to reduce the incidence of neonatal infections when administered intrapartum, and to improve survival when administered in infancy. Questions remain regarding whether azithromycin can prevent stillbirths, and regarding the optimal strategy for the delivery of azithromycin to pregnant women and their infants. METHODS AND ANALYSIS: Sauver avec l'Azithromycine en Traitant les Femmes Enceintes et les Enfants (SANTE) is a 2×2 factorial, individually randomised, placebo-controlled, double-masked trial in rural Mali. The primary aims are: (1A) to assess the efficacy of antenatal and intrapartum azithromycin on a composite outcome of stillbirths and infant mortality through 6-12 months and (1B) to assess the efficacy of azithromycin administered concurrently with the first and third doses of pentavalent vaccines (Penta-1/3) on infant mortality through 6-12 months. Pregnant participants (n=49 600) and their infants are randomised 1:1:1:1 to one of four treatment arms: (1) mother and infant receive azithromycin, (2) mother and infant receive placebo, (3) mother receives azithromycin and infant receives placebo or (4) mother receives placebo and infant receives azithromycin. Pregnant participants receive three single 2 g doses: two antepartum and one intrapartum. Infants receive a single 20 mg/kg dose at the Penta-1 and 3 visits. An additional cohort of 12 000 infants is recruited at the Penta-1 visit and randomised 1:1 to receive azithromycin or placebo at the same time points. The SANTE trial will inform guidelines and policies regarding the administration of antenatal and infant azithromycin using routine healthcare delivery platforms. ETHICS AND DISSEMINATION: This trial was approved by the Institutional Review Board at the University of Maryland School of Medicine (Protocol #HP-00084242) and the Faculté de Médecine et d'Odonto-Stomatologie in Mali. The findings of this trial will be published in open access peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03909737.

Reduction of No-Show Rate for New Patients in a Pediatric Neurology Clinic.

BACKGROUND: The rate of patients not keeping their appointments at our children's hospital outpatient pediatric neurology clinic (no-shows) was high. We conducted a quality improvement project to reduce no-show rates and improve operational efficiency. Specifically, we aimed to decrease the new patient no-show mean rate from 7% to 4% at the main campus and from 17% to 12% at the south campus. METHODS: After reviewing the previous literature on this topic and institutional data, we used the simplified failure mode and effects analysis (sFMEA) to identify the key drivers. Of the patients at the main campus who failed to keep their appointment, 84% had not confirmed their appointment. Errors in inpatient/family contact information, limited use of the electronic patient portal, and miscommunication were other key drivers identified. Three Plan-Do-Study-Act (PDSA) cycles were completed over seven months. The key interventions we implemented were bidirectional text triage, telephone reminders, and promoting the use of the electronic patient portal. A run chart was used to assess the results of these interventions. RESULTS: A statistically significant shift was noted in the run chart for the median rate of no-shows, which declined from 7% to 4% at the main campus and 17% to 10% at the south campus. CONCLUSION: We were able to successfully reduce no-shows among new patients in the neurology clinic. The limitations of our study include unknown external factors, the potential impact of COVID-19, and the brief length of the study.

Design and fabrication of a low-cost and programmable dip coating machine.

Dip coaters provide a compact device that can be used to obtain uniform and consistent coatings on a substrate. This article describes a less costly dip coater design with similar specifications to other dip coaters. The machine consists of a NEMA 23 stepper motor, touch screen display, and acrylic housing to protect the substrate from dust. The machine is programmed using Arduino IDE, allowing for easy changes to be made. The small size of the dip coater makes it ideal for use in labs. The majority of the components require no alterations after purchase, allowing for easy replacements and construction.

Serologic and Cytokine Profiles of Children with Concurrent Cerebral Malaria and Severe Malarial Anemia Are Distinct from Other Subtypes of Severe Malaria.

We used a protein microarray featuring Plasmodium falciparum field variants of a merozoite surface antigen to examine malaria exposure in Malian children with different severe malaria syndromes. Unlike children with cerebral malaria alone or severe malarial anemia alone, those with concurrent cerebral malaria and severe malarial anemia had serologic responses demonstrating a broader prior parasite exposure pattern than matched controls with uncomplicated disease. Comparison of levels of malaria-related cytokines revealed that children with the concurrent phenotype had elevated levels of interleukin (IL)-6, IL-8, and IL-10. Our results suggest that the pathophysiology of this severe subtype is unique and merits further investigation.

Malian adults maintain serologic responses to virulent PfEMP1s amid seasonal patterns of fluctuation.

Plasmodium falciparum erythrocyte membrane protein-1s (PfEMP1s), diverse malaria proteins expressed on the infected erythrocyte surface, play an important role in pathogenesis, mediating adhesion to host vascular endothelium. Antibodies to particular non-CD36-binding PfEMP1s are associated with protection against severe disease. We hypothesized that given lifelong P. falciparum exposure, Malian adults would have broad PfEMP1 serorecognition and high seroreactivity levels during follow-up, particularly to non-CD36-binding PfEMP1s such as those that attach to endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1). Using a protein microarray, we determined serologic responses to 166 reference PfEMP1 fragments during a dry and subsequent malaria transmission season in Malian adults. Malian adult sera had PfEMP1 serologic responses throughout the year, with decreased reactivity to a small subset of PfEMP1 fragments during the dry season and increases in reactivity to a different subset of PfEMP1 fragments during the subsequent peak malaria transmission season, especially for intracellular PfEMP1 domains. For some individuals, PfEMP1 serologic responses increased after the dry season, suggesting antigenic switching during asymptomatic infection. Adults were more likely to experience variable serorecognition of CD36-binding PfEMP1s than non-CD36-binding PfEMP1s that bind EPCR or ICAM-1, which remained serorecognized throughout the year. Sustained seroreactivity to non-CD36-binding PfEMP1s throughout adulthood amid seasonal fluctuation patterns may reflect underlying protective severe malaria immunity and merits further investigation.

Measuring the Barriers to Adherence With Neurology Clinic Appointments for Children With Epilepsy: A Pilot Study.

INTRODUCTION: Epilepsy is one of the most common neurological disorders in children. Missed appointments reflect missed opportunity to provide care for children with epilepsy. The objective of this study was to identify social determinants of health (SDH) and other factors associated with missed appointments in children with epilepsy and measure the relation between missed appointments and frequency of emergency room (ER) visits and inpatient admissions. METHODS: This was a prospective study conducted in the neurology division at a level 4 epilepsy center. Children (0 to < 18 years of age) with a diagnosis of epilepsy were included and a semi-structured questionnaire was provided to the families. Patients with 2 or more missed neurology clinic appointments in the previous year ("study group", n = 36) were compared to those with 1 or zero missed appointments ("control group", n = 49). A comparison of the clinical characteristics, emergency room visits and hospitalizations in the past year as well as SDH was performed. Statistical analysis was performed using SPSS and p < 0.05 was considered significant. RESULTS: The mean age, gender distribution and presence of medical refractoriness were comparable between the 2 groups. Families in the study group reported a higher likelihood of having to make special work arrangements for clinic appointments. Children in the study group were noted to have a significantly higher frequency of single mother households, presence of public insurance, father not graduating from high school and household income less than 50,000 dollars. Within the preceding year, children in the study group were noted to have a higher frequency of visits to the emergency department as well as 6 times higher likelihood of inpatient hospitalization for seizures. CONCLUSIONS: Social determinants of health play an important role in determining adherence with neurology clinic visits in children with epilepsy. Children with more missed appointments are likely to have a higher frequency of visits to the emergency department as well as a higher incidence of hospitalization for seizures. Identification of high-risk families and implementation of early interventions may improve adherence to office visits and decrease emergency room visits and hospitalization for seizures.

Vaccine-related major cutaneous reaction size correlates with cellular-mediated immune responses after tularaemia immunisation.

OBJECTIVES: Francisella tularensis, the causative agent of tularaemia, is an exceptionally infectious bacterium, potentially fatal for humans if left untreated and with the potential to be developed as a bioweapon. Both natural infection and live-attenuated vaccine strain (LVS) confer good protection against tularaemia. LVS vaccination is traditionally administered by scarification, and the formation of a cutaneous reaction or take at the vaccination site is recognised as a clinical correlate of protection. Although previous studies have suggested that high antibody titres following vaccination might serve as a useful surrogate marker, the immunological correlates of protection remain unknown. METHODS: We investigated the host T-cell-mediated immune (T-CMI) responses elicited following immunisation with LVS vaccine formulated by the DynPort Vaccine Company (DVC-LVS) or the United States Army Medical Research Institute of Infectious Diseases (USAMRIID-LVS). We compared T-CMI responses prompted by these vaccines and correlated them with take size. RESULTS: We found that both LVS vaccines elicited similar T-CMI responses. Interestingly, take size associated with the T cells' ability to proliferate, secrete IFN-γ and mobilise degranulation, suggesting that these responses play an essential role in tularaemia protection. CONCLUSIONS: These results renew the appreciation for vaccination through the scarification as a prime route of inoculation to target pathogens driving specific T-CMI responses and provide further evidence that T-CMI plays a role in protection from tularaemia.

Epitope-Specific Antibody Responses to a Plasmodium falciparum Subunit Vaccine Target in a Malaria-Endemic Population.

Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data.

Microarray analyses reveal strain-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 variants following natural infection and vaccination.

Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.

Thermal biology and swimming performance of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus).

Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) are two commercially important marine fishes impacted by both overfishing and climate change. Increasing ocean temperatures are affecting the physiology of these species and causing changes in distribution, growth, and maturity. While the physiology of cod has been well investigated, that of haddock has received very little attention. Here, we measured the metabolic response to increasing temperatures, as well as the critical thermal maximum (CTmax), of cod acclimated to 8 and 12 °C and haddock acclimated to 12 °C. We also compared the swimming performance (critical swimming speed, U crit) of cod and haddock at 12 °C, as well as the U crit of 12 °C-acclimated cod acutely exposed to a higher-than-optimal temperature (16 °C). The CTmax for cod was 21.4 and 23.0 °C for 8- and 12 °C-acclimated fish, respectively, whereas that for the 12 °C-acclimated haddock was 23.9 °C. These values were all significantly different and show that haddock are more tolerant of high temperatures. The aerobic maximum metabolic rate (MMR) of swimming cod remained high at 16 °C, suggesting that maximum oxygen transport capacity was not limited at a temperature above optimal in this species. However, signs of impaired swimming (struggling) were becoming evident at 16 °C. Haddock were found to reach a higher U crit than cod at 12 °C (3.02 vs. 2.62 body lengths s-1, respectively), and at a lower MMR. Taken together, these results suggest that haddock perform better than cod in warmer conditions, and that haddock are the superior swimmer amongst the two species.

Serologic responses to the PfEMP1 DBL-CIDR head structure may be a better indicator of malaria exposure than those to the DBL-α tag.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α "tag"). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or "parent" domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). METHODS: A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. RESULTS: Malian serologic responses to a PfEMP1's DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. CONCLUSIONS: Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.

The acute and incremental thermal tolerance of Atlantic cod (Gadus morhua) families under normoxia and mild hypoxia.

Given climate change projections, the limited ability of fish reared in sea-cages to behaviourally thermoregulate, and that thermal tolerance may be heritable, studies that examine family-related differences in upper thermal tolerance are quite relevant to the aquaculture industry. Thus, we investigated the upper thermal tolerance of 15 Atlantic cod (Gadus morhua L.) families by challenging them with acute (2 °C h-1) and incremental (1 °C every 4 days) temperature increases (CTmax and ITmax tests, respectively) under normoxia (~ 100% air saturation) and mild hypoxia (~ 75% air sat.). The cod's CTmax was 22.5 ±â€¯0.1 °C (mean ±â€¯S.E.) during normoxia and 21.8 ±â€¯0.1 °C during hypoxia (P < 0.001); and these two CTmax values were significantly correlated across families. In both the normoxic and hypoxic ITmax tests, feed intake fell by ~50% between 17 and 18 °C, and stopped entirely by 21 °C. No mortalities were observed under 20 °C in the normoxic and hypoxic ITmax tests, and the ITmax value was ~21.7 °C in both groups. Differences in the upper thermal tolerance between families were only observed in the CTmax experiment. No correlation was found between the specific growth rate and the CTmax of the families. Further, no correlation existed between CTmax and ITmax. This study is the first to compare the thermal tolerance of fish families to both CTmax and ITmax challenges, and the data: 1) suggest that the Atlantic cod is quite tolerant of acute (i.e., hours) or short-term (i.e., weeks) exposure to high water temperatures (i.e., up to 20 °C); 2) indicate that it might be difficult to select fish with higher ITmax values; and 3) question the relevance of CTmax for selecting fish that are destined for sea-cages where temperatures slowly warm over the summer.

Antibodies to Peptides in Semiconserved Domains of RIFINs and STEVORs Correlate with Malaria Exposure.

The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria.IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.

Clinical, Virologic, and Immunologic Characteristics of Zika Virus Infection in a Cohort of US Patients: Prolonged RNA Detection in Whole Blood.

BACKGROUND: Clinical, virologic, and immunologic characteristics of Zika virus (ZIKV) infections in US patients are poorly defined. METHODS: US subjects with suspected ZIKV infection were enrolled. Clinical data and specimens were prospectively collected for ZIKV RNA detection and serologic and cellular assays. Confirmed ZIKV infection (cases) and ZIKV-negative (controls) subjects were compared. Dengue-experienced and dengue-naïve cases were also compared. RESULTS: We enrolled 45 cases and 14 controls. Commonly reported symptoms among cases and controls were maculopapular rash (97.8% and 81.8%), fatigue (86.7% and 81.8%), and arthralgia (82.2% and 54.5%), respectively. The sensitivity (94%) and duration of infection detection (80% positivity at 65-79 days after disease onset) by polymerase chain reaction were highest in whole-blood specimens. ZIKV-neutralizing antibodies had a half-life of 105 days and were significantly higher in dengue virus-experienced cases than naïve ones (P = .046). In intracellular cytokine staining assays, the ZIKV proteins targeted most often by peripheral blood mononuclear cells from cases were structural proteins C and E for CD4+ T cells and nonstructural proteins NS3, NS5, and NS4B for CD8+ T cells. CONCLUSIONS: ZIKV RNA detection was more frequent and prolonged in whole-blood specimens. Immunoglobulin G (IgG) and neutralizing antibodies, but not IgM, were influenced by prior dengue infection. Robust cellular responses to E and nonstructural proteins have potential vaccine development implications.

Cross-Platform Comparison of Computer-assisted Image Analysis Quantification of In Situ mRNA Hybridization in Investigative Pathology.

Although availability of automated platforms has proliferated, there is no standard practice for computer-assisted generation of scores for mRNA in situ hybridization (ISH) visualized by brightfield microscopic imaging on tissue sections. To address this systematically, an ISH for peptidylprolyl isomerase B (PPIB) (cyclophilin B) mRNA was optimized and applied to a tissue microarray of archival non-small cell lung carcinoma cases, and then automated image analysis for PPIB was refined across 4 commercially available software platforms. Operator experience and scoring results from ImageScope, HALO, CellMap, and Developer XD were systematically compared with each other and to manual pathologist scoring. Markup images were compared and contrasted for accuracy, the ability of the platform to identify cells, and the ease of visual assessment to determine appropriate interpretation. Comparing weighted scoring approaches using H-scores (Developer XD, ImageScope, and manual scoring) a correlation was observed (R value=0.7955), and association between the remaining 2 approaches (HALO and CellMap) was of similar value. ImageScope showed the highest R value in comparison with manual scoring (0.7377). Mean-difference plots showed that HALO produced the highest relative normalized values, suggesting higher relative sensitivity. ImageScope overestimated PPIB ISH signal at the high end of the range scores; however, this tendency was not observed in other platforms. HALO emerged with the highest number of favorable observations, no apparent systematic bias in score generation compared with the other methods, and potentially higher sensitivity to detect ISH. HALO may serve as a tool to empower teams of investigative pathology laboratory scientists to assist pathologists readily with quantitative scoring of ISH.

Do Deauville Scores Improve the Clinical Utility of End-of-Therapy FDG PET Scans for Pediatric Hodgkin Lymphoma?

OBJECTIVE: The purpose of this study was to evaluate the clinical utility of Deauville scores in interpretation of end-of-chemotherapy FDG PET scans. CONCLUSION: Deauville scores improve the clinical utility of end-of-chemotherapy PET, as evidenced by an increase in positive predictive value to 72.7% from 44.4% on the basis of report alone. The negative predictive value remains greater than 95%.

Does Extended PET Acquisition in PET/MRI Rectal Cancer Staging Improve Results?

OBJECTIVE: The purpose of this study was to determine if extended PET acquisition times in the pelvis during PET/MRI increase detection rates of potentially metastatic lymph nodes in patients with rectal cancer. MATERIALS AND METHODS: Our study was approved by the institutional review board of the University of California, San Francisco. Twenty-two patients with biopsy-proven rectal cancer underwent imaging via simultaneous 3-T time-of-flight PET/MRI, with seven undergoing two separate PET/MRI examinations, for a total of 29 studies. Each examination included both a whole-body PET/MRI and a dedicated pelvic PET/MRI with both 3- and 15-minute PET acquisitions for the pelvis. Three radiologists interpreted each examination with PET only, MRI only, then combined PET and MRI examinations, using all available images. Additionally, the 3- and 15-minute PET acquisitions of the pelvis were reviewed separately by a single radiologist. RESULTS: A total of 94 lymph nodes were identified as abnormal on PET, all with MRI anatomic correlates. Of these, 37 (39.4%) were seen only on the dedicated 15-minute acquisition. Fifty-seven (60.6%) nodes measured 5 mm or less, including 29 (30.9%) seen only on the 15-minute acquisition. Thirty-one (33.0%) nodes measured 5.1-10 mm, including eight (25.8%) seen only on the 15-minute acquisition. Of the 17 subjects imaged for initial staging, 11 (64.7%) were upstaged as a result of the increased PET acquisition time (10 from N1 to N2 and one from N0 to N1). CONCLUSION: Longer PET acquisition times during PET/MRI for rectal cancer increases the number of FDG-avid lymph nodes detected without increasing scan time.

Children with cerebral malaria or severe malarial anaemia lack immunity to distinct variant surface antigen subsets.

Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or "lacunae", in immunity to these Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children.