Modest heterologous protection after Plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial.

BACKGROUND: A highly efficacious vaccine is needed for malaria control and eradication. Immunization with Plasmodium falciparum NF54 parasites under chemoprophylaxis (chemoprophylaxis and sporozoite (CPS)-immunization) induces the most efficient long-lasting protection against a homologous parasite. However, parasite genetic diversity is a major hurdle for protection against heterologous strains. METHODS: We conducted a double-blind, randomized controlled trial in 39 healthy participants of NF54-CPS immunization by bites of 45 NF54-infected (n = 24 volunteers) or uninfected mosquitoes (placebo; n = 15 volunteers) against a controlled human malaria infection with the homologous NF54 or the genetically distinct NF135.C10 and NF166.C8 clones. Cellular and humoral immune assays were performed as well as genetic characterization of the parasite clones. RESULTS: NF54-CPS immunization induced complete protection in 5/5 volunteers against NF54 challenge infection at 14 weeks post-immunization, but sterilely protected only 2/10 and 1/9 volunteers against NF135.C10 and NF166.C8 challenge infection, respectively. Post-immunization plasma showed a significantly lower capacity to block heterologous parasite development in primary human hepatocytes compared to NF54. Whole genome sequencing showed that NF135.C10 and NF166.C8 have amino acid changes in multiple antigens targeted by CPS-induced antibodies. Volunteers protected against heterologous challenge were among the stronger immune responders to in vitro parasite stimulation. CONCLUSIONS: Although highly protective against homologous parasites, NF54-CPS-induced immunity is less effective against heterologous parasite clones both in vivo and in vitro. Our data indicate that whole sporozoite-based vaccine approaches require more potent immune responses for heterologous protection. TRIAL REGISTRATION: This trial is registered in clinicaltrials.gov, under identifier NCT02098590 .

BAFF and BAFF receptor levels correlate with B cell subset activation and redistribution in controlled human malaria infection.

Characteristic features of Plasmodium falciparum malaria are polyclonal B cell activation and an altered composition of the blood B cell compartment, including expansion of CD21(-)CD27(-) atypical memory B cells. BAFF is a key cytokine in B cell homeostasis, but its potential contribution to the modulation of the blood B cell pool during malaria remains elusive. In the controlled human malaria model (CHMI) in malaria-naive Dutch volunteers, we therefore examined the dynamics of BAFF induction and B cell subset activation and composition, to investigate whether these changes are linked to malaria-induced immune activation and, in particular, induction of BAFF. Alterations in B cell composition after CHMI closely resembled those observed in endemic areas. We further found distinct kinetics of proliferation for individual B cell subsets across all developmental stages. Proliferation peaked either immediately after blood-stage infection or at convalescence, and for most subsets was directly associated with the peak parasitemia. Concomitantly, plasma BAFF levels during CHMI were increased and correlated with membrane-expressed BAFF on monocytes and dendritic cells, as well as blood-stage parasitemia and parasite-induced IFN-γ. Correlating with increased plasma BAFF and IFN-γ levels, IgD(-)CD38(low)CD21(-)CD27(-) atypical B cells showed the strongest proliferative response of all memory B cell subsets. This provides unique evidence for a link between malaria-induced immune activation and temporary expansion of this B cell subset. Finally, baseline BAFF-R levels before CHMI were predictive of subsequent changes in proportions of individual B cell subsets. These findings suggest an important role of BAFF in facilitating B cell subset proliferation and redistribution as a consequence of malaria-induced immune activation.

Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity.

Volunteers immunized under chloroquine chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) develop complete, long-lasting protection against homologous sporozoite challenge. Chloroquine affects neither sporozoites nor liver-stages, but kills only asexual forms in erythrocytes once released from the liver into the circulation. Consequently, CPS immunization exposes the host to antigens from both preerythrocytic and blood stages, and induced immunity might target either of these stages. We therefore explored the life cycle stage specificity of CPS-induced protection. Twenty-five malaria-naïve volunteers were enrolled in a clinical trial, 15 of whom received CPS immunization. Five immunized subjects and five controls received a sporozoite challenge by mosquito bites, whereas nine immunized and five control subjects received an i.v. challenge with P. falciparum-infected erythrocytes. The latter approach completely bypasses preerythrocytic stages, enabling a direct comparison of protection against either life cycle stage. CPS-immunized subjects (13 of 14) developed anticircumsporozoite antibodies, whereas only one volunteer generated minimal titers against typical blood-stage antigens. IgG from CPS-immunized volunteers did not inhibit asexual blood-stage growth in vitro. All CPS-immunized subjects (5 of 5) were protected against sporozoite challenge. In contrast, nine of nine CPS-immunized subjects developed parasitemia after blood-stage challenge, with identical prepatent periods and blood-stage multiplication rates compared with controls. Intravenously challenged CPS-immunized subjects showed earlier fever and increased plasma concentrations of inflammatory markers D-dimer, IFN-γ, and monokine induced by IFN-γ than i.v. challenged controls. The complete lack of protection against blood-stage challenge indicates that CPS-induced protection is mediated by immunity against preerythrocytic stages. However, evidence is presented for immune recognition of P. falciparum-infected erythrocytes, suggesting memory responses unable to generate functional immunity.

Controlled human malaria infection trials: How tandems of trust and control construct scientific knowledge.

Controlled human malaria infections are clinical trials in which healthy volunteers are deliberately infected with malaria under controlled conditions. Controlled human malaria infections are complex clinical trials: many different groups and institutions are involved, and several complex technologies are required to function together. This functioning together of technologies, people, and institutions is under special pressure because of potential risks to the volunteers. In this article, the authors use controlled human malaria infections as a strategic research site to study the use of control, the role of trust, and the interactions between trust and control in the construction of scientific knowledge. The authors argue that tandems of trust and control play a central role in the successful execution of clinical trials and the construction of scientific knowledge. More specifically, two aspects of tandems of trust and control will be highlighted: tandems are sites where trust and control coproduce each other, and tandems link the personal, the technical, and the institutional domains. Understanding tandems of trust and control results in setting some agendas for both clinical trial research and science and technology studies.

Plasmodium falciparum Infection of Human Volunteers Activates Monocytes and CD16+ Dendritic Cells and Induces Upregulation of CD16 and CD1c Expression.

Antigen-presenting cells (APCs) are key players in the induction and regulation of immune responses. In Plasmodium falciparum malaria, determination of which cells and pathways are activated in the network of APCs remains elusive. We therefore investigated the effects of a controlled human malaria infection in healthy, malaria-naive volunteers on the subset composition and activation status of dendritic cells (DCs) and monocytes. While subsets of monocytes increased in frequency during blood-stage infection, DC frequencies remained largely stable. Activation markers classically associated with peptide presentation to and priming of αßT cells, HLA-DR and CD86, were upregulated in monocytes and inflammatory CD16 myeloid DCs (mDCs) but not in the classical CD1c, BDCA2, or BDCA3 DC subsets. In addition, these activated APC subsets showed increased expression of CD1c, which is involved in glycolipid antigen presentation, and of the immune complex binding Fcγ receptor III (CD16). Our data show that P. falciparum asexual parasites do not activate classical DC subsets but instead activate mainly monocytes and inflammatory CD16 mDCs and appear to prime alternative activation pathways via induction of CD16 and/or CD1c. Changes in expression of these surface molecules might increase antigen capture and enhance glycolipid antigen presentation in addition to the classical major histocompatibility complex class II (MHC-II) peptide presentation and thereby contribute to the initiation of T-cell responses in malaria. (This study has been registered at Clinicaltrials.gov under registration no. NCT01086917.).

Impact of malaria preexposure on antiparasite cellular and humoral immune responses after controlled human malaria infection.

To understand the effect of previous malaria exposure on antiparasite immune responses is important for developing successful immunization strategies. Controlled human malaria infections (CHMIs) using cryopreserved Plasmodium falciparum sporozoites provide a unique opportunity to study differences in acquisition or recall of antimalaria immune responses in individuals from different transmission settings and genetic backgrounds. In this study, we compared antiparasite humoral and cellular immune responses in two cohorts of malaria-naive Dutch volunteers and Tanzanians from an area of low malarial endemicity, who were subjected to the identical CHMI protocol by intradermal injection of P. falciparum sporozoites. Samples from both trials were analyzed in parallel in a single center to ensure direct comparability of immunological outcomes. Within the Tanzanian cohort, we distinguished one group with moderate levels of preexisting antibodies to asexual P. falciparum lysate and another that, based on P. falciparum serology, resembled the malaria-naive Dutch cohort. Positive P. falciparum serology at baseline was associated with a lower parasite density at first detection by quantitative PCR (qPCR) after CHMI than that for Tanzanian volunteers with negative serology. Post-CHMI, both Tanzanian groups showed a stronger increase in anti-P. falciparum antibody titers than Dutch volunteers, indicating similar levels of B-cell memory independent of serology. In contrast to the Dutch, Tanzanians failed to increase P. falciparum-specific in vitro recall gamma interferon (IFN-γ) production after CHMI, and innate IFN-γ responses were lower in P. falciparum lysate-seropositive individuals than in seronegative individuals. In conclusion, positive P. falciparum lysate serology can be used to identify individuals with better parasite control but weaker IFN-γ responses in circulating lymphocytes, which may help to stratify volunteers in future CHMI trials in areas where malaria is endemic.

Studying the effect of chloroquine on sporozoite-induced protection and immune responses in Plasmodium berghei malaria.

BACKGROUND: Sporozoite immunization of animals and humans under a chemo-prophylactic cover of chloroquine (CPS-CQ) efficiently induces sterile protection against malaria. In humans, CPS-CQ is strikingly more efficient than immunization with radiation attenuated sporozoites (RAS), raising the hypothesis that this might be partially due to CQ. Chloroquine, an established anti-malarial drug, is also well known for its immune modulating properties including improvement of cross-presentation. The aim of this study was to investigate whether co-administration of CQ during sporozoite immunization improves cellular responses and protective efficacy in Plasmodium berghei models. METHODS: A number of experiments in selected complimentary P. berghei murine models in Balb/cByJ and C57BL/6j mice was performed. First, the effect of CQ administration on the induction of protection and immune responses by RAS immunization was studied. Next, the effect of CQ on the induction of circumsporozoite (CS) protein-specific CD8(+) T cells by immunization with P. berghei parasites expressing a mutant CS protein was investigated. Finally, a direct comparison of CPS-CQ to CPS with mefloquine (MQ), an anti-malarial with little known immune modulating effects, was performed. RESULTS: When CQ was co-administered during immunization with graded numbers of RAS, this did not lead to an increase in frequencies of total memory CD8(+) T cells or CS protein-specific CD8(+) T cells. Also parasite-specific cytokine production and protection remained unaltered. Replacement of CQ by MQ for CPS immunization resulted in significantly reduced percentages of IFNγ producing memory T cells in the liver (p = 0.01), but similar protection. CONCLUSIONS: This study does not provide evidence for a direct beneficial effect of CQ on the induction of sporozoite-induced immune responses and protection in P. berghei malaria models. Alternatively, the higher efficiency of CPS compared to RAS might be explained by an indirect effect of CQ through limiting blood-stage exposure after immunization or to increased antigen exposure and, therefore, improved breadth of the immune response.

Cytotoxic markers associate with protection against malaria in human volunteers immunized with Plasmodium falciparum sporozoites.

BACKGROUND: Immunization of healthy volunteers by bites from Plasmodium falciparum-infected mosquitoes during chloroquine chemoprophylaxis (hereafter, chemoprophylaxis and sporozoites [CPS] immunization) induces sterile protection against malaria. CPS-induced protection is mediated by immunity against pre-erythrocytic stages, presumably at least partially by cytotoxic cellular responses. We therefore aimed to investigate the association of CPS-induced cytotoxic T-cell markers with protection. METHODS: In a double-blind randomized controlled trial, we performed dose titration of CPS immunization followed by homologous challenge infection in 29 subjects. Immune responses were assessed by in vitro restimulation of peripheral blood mononuclear cells and flow cytometry. RESULTS: Dose-dependent complete protection was obtained in 4 of 5 volunteers after immunization with bites from 45 P. falciparum-infected mosquitoes, in 8 of 9 volunteers with bites from 30, and in 5 of 10 volunteers with bites from 15 (odds ratio [OR], 5.0; 95% confidence interval [CI], 1.5-17). Completely protected subjects had significantly higher proportions of CD4 T cells expressing the degranulation marker CD107a (OR, 8.4; 95% CI, 1.5-123; P = .011) and CD8 cells producing granzyme B (OR, 11; 95% CI, 1.9-212; P = .004) after P. falciparum restimulation. CONCLUSIONS: These data underline the efficiency of CPS immunization to induce sterile protection and support a possible role for cytotoxic CD4 and CD8 T-cell responses in pre-erythrocytic immunity. CLINICAL TRIALS REGISTRATION: NCT01218893.

Memory B-cell and antibody responses induced by Plasmodium falciparum sporozoite immunization.

BACKGROUND: Immunization of healthy volunteers during receipt of chemoprophylaxis with Plasmodium falciparum sporozoites (CPS-immunization) induces sterile protection from malaria. Antibody responses have long been known to contribute to naturally acquired immunity against malaria, but their association with sterile protection after whole sporozoite immunization is not well established. We therefore studied the induction and kinetics of malaria parasite antigen-specific antibodies and memory B-cells (MBCs) during CPS-immunization and their correlation with protection from challenge infection. METHODS: We assessed humoral reactivity to 9 antigens representing different stages of the life cycle of P. falciparum by performing standardized MBC enzyme-linked immunospot and enzyme-linked immunosorbent assays on peripheral blood mononuclear cells and plasma samples from 38 Dutch volunteers enrolled in 2 randomized controlled clinical trials. RESULTS: MBCs and antibodies recognizing pre-erythrocytic and cross-stage antigens were gradually acquired during CPS-immunization. The magnitude of these humoral responses did not correlate with protection but directly reflected parasite exposure in CPS-immunization and challenge. CONCLUSIONS: Humoral responses to the malarial antigens circumsporozoite protein, liver-stage antigen-1, apical membrane antigen-1, and merozoite surface protein-1 do not to predict protection from challenge infection but can be used as sensitive marker of recent parasite exposure. CLINICAL TRIALS REGISTRATION: NCT01236612 and NCT01218893.

Sporozoite immunization of human volunteers under chemoprophylaxis induces functional antibodies against pre-erythrocytic stages of Plasmodium falciparum.

BACKGROUND: Long-lasting and sterile protective immunity against Plasmodium falciparum can be achieved by immunization of malaria-naive human volunteers under chloroquine prophylaxis with sporozoites delivered by mosquito bites (CPS-immunization). Protection is mediated by sporozoite/liver-stage immunity. In this study, the capacity of CPS-induced antibodies to interfere with sporozoite functionality and development was explored. METHODS: IgG was purified from plasma samples obtained before and after CPS-immunization from two separate clinical trials. The functionality of these antibodies was assessed in vitro in gliding and human hepatocyte traversal assays, and in vivo in a human liver-chimeric mouse model. RESULTS: Whereas pre-treatment of sporozoites with 2 mg/ml IgG in the majority of the volunteers did not have an effect on in vitro sporozoite gliding motility, CPS-induced IgG showed a distinct inhibitory effect in the sporozoite in vitro traversal assay. Pre-treatment of P. falciparum sporozoites with post-immunization IgG significantly inhibited sporozoite traversal through hepatocytes in 9/9 samples when using 10 and 1 mg/ml IgG, and was dose-dependent, resulting in an average 16% and 37% reduction with 1 mg/ml IgG (p = 0.003) and 10 mg/ml IgG (p = 0.002), respectively. In vivo, CPS-induced IgG reduced liver-stage infection and/or development after a mosquito infection in the human liver-chimeric mouse model by 91.05% when comparing 11 mice receiving post-immunization IgG to 11 mice receiving pre-immunization IgG (p = 0.0008). CONCLUSIONS: It is demonstrated for the first time that CPS-immunization induces functional antibodies against P. falciparum sporozoites, which are able to reduce parasite-host cell interaction by inhibiting parasite traversal and liver-stage infection. These data highlight the functional contribution of antibody responses to pre-erythrocytic immunity after whole-parasite immunization against P. falciparum malaria.

The inclusion of children and adolescents in tuberculosis diagnostic development and evaluation-a consensus statement.

The diagnosis of paediatric tuberculosis remains a challenge due to the non-specificity of symptoms and the paucibacillary nature of tuberculosis in children. However, in the development of new tuberculosis diagnostics, the unique needs of children and adolescents are rarely considered in the design process, with delays in evaluation and approval. No clear guidance is available on when and how to include children and adolescents in tuberculosis diagnostic development and evaluation. To address this gap, we conducted a Delphi consensus process with 42 stakeholders, including one qualitative and two quantitative rounds. Consensus was achieved on 20 statements, with agreement that the needs and perspectives of children, adolescents, and their caregivers should be incorporated throughout diagnostic design and evaluation. Opportunities exist for the early use of well characterised samples and prospective enrolment of children and adolescents in tuberculosis diagnostic evaluation, with consideration of the type of test, expected benefit, and potential risks. Pathogen-based tests might be initially optimised and assessed in adults and adolescents, but parallel evaluation in children is needed for host-based tests. Late-stage evaluation and implementation studies should examine combination testing and integration into clinical algorithms. The statements support collaboration between developers, researchers, regulators, and users to widen and accelerate the diagnostic pipeline for paediatric tuberculosis.

Malaria and tuberculosis co-infection-a review.

Malaria and tuberculosis remain highly prevalent infectious diseases and continue to cause significant burden worldwide. Endemic regions largely overlap, and co-infections are expected to occur frequently. Surprisingly, malaria-tuberculosis co-infection is relatively understudied. Malaria has long been known to have immunomodulatory effects, for example resulting in reduced vaccination responses against some pathogens, and it is conceivable that this also plays a role if co-infection occurs. Data from animal studies indeed suggest clinically important effects of malaria-tuberculosis co-infection on the immune responses with potential consequences for the pathophysiology and clinical course of both infections. Specifically, rodent studies consistently show reduced control of mycobacteria during malaria infection. Although the underlying immunological mechanisms largely remain unclear, an altered balance between pro- and anti-inflammatory responses may play a role. Some observations in humans also support the hypothesis that malaria infection skews the immune responses against tuberculosis, but data are limited. Further research is needed to unravel the underlying immunological mechanisms and delineate possible implications of malaria-tuberculosis co-infection for clinical practice.

Intracranial Empyema in Children: A Single-center Retrospective Case Series.

We conducted a retrospective, observational study of 42 children with intracranial empyema admitted to a pediatric neurosurgical center over a 9-year period. Intracranial empyema is rare, but causes significant morbidity and mortality. Twenty-eight cases had neurosurgical source control, more commonly for subdural collections. Streptococcus anginosus group bacteria are important pathogens in subdural empyema, whose isolation predicts more complicated postoperative courses.

Kikuchi Disease in Children: Case Report and Review of the Literature.

BACKGROUND: Kikuchi disease (KD) is a rare and generally benign condition of uncertain etiology that presents with nonspecific symptoms including fever and cervical lymphadenopathy. Clinical presentations can vary. Here, we present an atypical case of KD in a 10-year-old girl, as well as an updated literature review of the clinical presentation, laboratory features and management of KD in children. METHODS: Studies (published up until February 2020) were identified through searches of PubMed using the following search items: Kikuchi-Fujimoto disease or histiocytic necrotizing lymphadenitis or Kikuchi disease. Our primary search resulted in 1117 publications. A total of 34 publications with a total of 670 patients were included in the final analysis. RESULTS: All children present with lymphadenopathy. Almost all (96.3%) have cervical lymphadenopathy. Fever is recorded in the majority of children (77.1%). Analysis of laboratory features found that the majority of children have leukopenia (56.0%) and a raised erythrocyte sedimentation rate (56.0%). Over 30% have a raised C-reactive protein and anemia. Other features such as leukocytosis, thrombocytopenia and antinuclear antibodies positivity are less common. KD is mostly self-limiting, but steroids, hydroxychloroquine and intravenous immunoglobulin are used in protracted courses. Their efficacy has yet to be established in clinical trials. CONCLUSIONS: The presentation of KD is variable, and there is no specific set of symptoms or laboratory features that reliably establishes the diagnosis. Thus, histopathology is crucial. Definitive evaluation and establishment of effective treatments will require future prospective research studies for a more comprehensive description of the clinical course and effects of treatment. Given the rarity of the disease, this will have to be performed in collaborative consortia.

Screening for Immunodeficiencies in Children With Invasive Pneumococcal Disease: Six-year Experience From a UK Children's Hospital.

BACKGROUND: A previous study showed that investigation of children with invasive pneumococcal disease (IPD) revealed an immunodeficiency in up to 10% of cases. Following this report, we implemented a protocol to investigate children with IPD, to assess the proportion with an immunodeficiency in our setting. METHODS: We retrospectively identified patients who presented with IPD from January 2015 to November 2020 and collected data from medical records. Immunological investigations included complement C3 and C4 levels, classical and alternative pathway complement function, IgG, IgA and IgM levels, specific IgG levels (H. influenza B, tetanus and pneumococcal serotypes), peripheral blood film, lymphocyte subsets, and CD62L-shedding upon activation with Toll-like receptor-agonists in selected cases. RESULTS: We identified a total of 68 children with IPD, with a mortality of 6%. Immunological investigations were performed in 51 children. Four children (8%) had abnormal findings that were deemed of clinical significance. Two children had complement deficiencies (Factor I and C2 deficiency), one child had specific antibody deficiency, and another child had low IgM, low NK-cells and poor persistence of serotype-specific anti-pneumococcal IgG concentrations. Of the 17 children with IPD who were not tested for immunodeficiencies, 4 died and four had possible explanations for the infection. CONCLUSIONS: We identified clinically relevant abnormal immunological findings in 4/51 (8%) of children with IPD. Our results support the recommendation to perform immunological investigations in children with IPD, since this might reveal underlying immunodeficiencies, allowing for necessary preventive measures and close follow-up.

A guide to vaccinology: from basic principles to new developments.

Immunization is a cornerstone of public health policy and is demonstrably highly cost-effective when used to protect child health. Although it could be argued that immunology has not thus far contributed much to vaccine development, in that most of the vaccines we use today were developed and tested empirically, it is clear that there are major challenges ahead to develop new vaccines for difficult-to-target pathogens, for which we urgently need a better understanding of protective immunity. Moreover, recognition of the huge potential and challenges for vaccines to control disease outbreaks and protect the older population, together with the availability of an array of new technologies, make it the perfect time for immunologists to be involved in designing the next generation of powerful immunogens. This Review provides an introductory overview of vaccines, immunization and related issues and thereby aims to inform a broad scientific audience about the underlying immunological concepts.

Gut mucosal cell damage in meningococcal sepsis in children: relation with clinical outcome.

OBJECTIVE: The pathophysiological sequelae of meningococcal sepsis are mainly caused by deregulated microvasculature function, leading to impaired tissue blood flow. Because mature enterocytes are known to be susceptible to altered perfusion, we aimed to investigate: (1) the development of enterocyte damage; and (2) the relation between enterocyte damage and severity of disease and outcome in children with meningococcal sepsis. DESIGN: Retrospective human study. SETTING: Pediatric intensive care unit at a university hospital. PATIENTS: Nineteen consecutive children with meningococcal sepsis were studied during their pediatric intensive care unit stay. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS Circulating levels of intestinal fatty acid binding protein, a small cytosolic protein constitutively present in mature enterocytes and released on cell injury, were assessed. Severity of disease was represented by meningococcal-specific Rotterdam Score, generic Pediatric Risk of Mortality II score, and circulating interleukin-6. Clinical outcome was measured by length of pediatric intensive care unit stay and number of ventilator days. Highest plasma intestinal fatty acid binding protein values were measured on pediatric intensive care unit stay admission. At the time of admission, eight of 19 patients had higher intestinal fatty acid binding protein plasma levels than the upper reference limit of 30 healthy volunteers. In all survivors, intestinal fatty acid binding protein levels declined to normal values within 12 hrs after starting intensive treatment, whereas the three nonsurvivors maintained elevated intestinal fatty acid binding protein plasma levels. A significant correlation was found among intestinal fatty acid binding protein and Rotterdam Score, Pediatric Risk of Mortality II, interleukin-6 at admission (Spearman's r = 0.402, p = .006; r = 0.243, p = .045; r = 0.687, p < .001, respectively). Next, a significant correlation was found between intestinal fatty acid binding protein and clinical outcome. CONCLUSIONS: Elevated plasma intestinal fatty acid binding protein is found in eight of 19 children with severe pediatric intensive care unit stay at the time of clinical presentation, suggesting the presence of enterocyte damage. Furthermore, prolonged enterocyte damage is found in nonsurvivors. Further studies are needed to clarify the potential role for assessment of plasma intestinal fatty acid binding protein in monitoring treatment of pediatric intensive care unit stay.

Sex Assignment and Diagnostics in Infants with Ambiguous Genitalia - A Single-Center Retrospective Study.

Ambiguous genitalia affect 1 in 5,000 live births. Diagnostic procedures can be time-consuming, and often the etiology cannot be established in this group of individuals with differences/disorders of sex development (DSD). We aimed to evaluate the clinical presentation, sex assignment, and diagnostic workup in these patients. In this retrospective observational study, we included infants who presented with ambiguous genitalia from 2006 to 2016 at the Radboudumc (Radboud University Medical Center) DSD expert center. Relevant data were collected from patient records. Sixty-two 46,XY and fourteen 46,XX individuals were included. Sex was assigned in the first days of life and based on the combination of presence or absence of a uterus on ultrasound, AMH level, palpable gonads, and the karyotype (corresponded in 96% of the patients). In 86% of the 46,XX DSD subjects, a diagnosis was made, whereas in only 15/62 (24%) of the 46,XY DSD individuals, etiology was determined. In 52 individuals, genetic testing was performed resulting in a diagnosis in 24 patients (46%). AMH, hCG-stimulated testosterone, and dihydrotestosterone levels contributed to determining etiology, whilst basal testosterone and basal dihydrotestosterone did not. Establishing a diagnosis in infants with ambiguous genitalia is complex and challenging; this study aids to enhance this process and improve current practice.