Clinical and Biochemical Features of Hypopituitarism Among Brazilian Children With Zika Virus-Induced Microcephaly.

Importance: The Zika virus infects progenitor neuron cells, disrupts cerebral development, and, in mice, drives hypothalamic defects. Patients with microcephaly caused by congenital Zika infection present with midline cerebral defects, which may result in hypopituitarism. Objective: To analyze postnatal growth and the presence of clinical and biochemical features associated with hypopituitarism in children with congenital Zika infections. Design, Setting, and Participants: In this prospective cohort study at 2 public referral hospitals in Bahia, Brazil, specializing in the treatment of congenital Zika infection, clinical data and growth parameters of 65 patients with the infection were evaluated. Data were analyzed from April 2017 through July 2018. Exposure: Congenital Zika infection. Main Outcomes and Measures: Length, weight, and head circumference were measured at birth and during follow up (ie, at 27 months of life) for each patient. Basal levels of free thyroxine, thyrotropin, cortisol, corticotropin, prolactin, insulin-like growth factor 1, insulin-like growth factor binding protein 3, urine and plasma osmolality, electrolytes, glucose, and insulin were evaluated at the age of 26 months to 28 months. All patients underwent central nervous system computed tomography scans and ophthalmic and otoacoustic evaluations at the time of this investigation or had done so previously. Results: Among 65 patients (38 [58.4%] male; median [interquartile range] age at enrollment, 27 [26-28] months), 61 patients presented with severe brain defects (93.8%), including corpus callosum agenesis or hypoplasia (ie, midline brain defects; 25 patients [38.5%]) and optic nerve atrophy (38 patients [58.5%]). Most patients presented with severe neurodevelopmental delay (62 of 64 patients [96.9%]). Past or present clinical signs of hypopituitarism were rare, occurring in 3 patients (4.6%). Severe microcephaly, compared with mild or moderate microcephaly, was associated with a shorter length by median (interquartile range) z score at birth (-1.9 [-2.5 to -1.0] vs -0.3 [-1.0 to 0]; P < .001), but this difference did not persist at 27 months (-1.6 [-2.3 to -0.3] vs -2.9 [-4.0 to -1.2]; P = .06). Growth hormone deficiency or hypothyroidism were not observed in any patients, and glucose and insulin levels were within reference ranges for all patients. Low cortisol levels (ie, below 3.9 µg/dL) were observed in 4 patients (6.2%). These 4 patients presented with low (ie, below 7.2 pg/mL) or inappropriately low (ie, below 30 pg/mL) corticotropin levels. Low corticotropin levels (ie, below 7.2 pg/mL) were observed in 6 patients (9.2%). Diabetes insipidus was evaluated in 21 patients; it was confirmed in 1 patient (4.8%) and suggested in 3 patients (14.3%). Conclusions and Relevance: This study found that congenital Zika infection with microcephaly was associated with midline brain defects and optic nerve atrophy. Children with congenital Zika infections presented with prenatal growth impairments with a lack of postnatal catch-up, as shown by persistent short length from birth until 27 months; these impairments were not associated with growth hormone deficiency. Patients also presented with severe developmental delay that was not associated with hypothyroidism, while central adrenal insufficiency and diabetes insipidus occurred in some patients.

Determinants of losses in the latent tuberculosis cascade of care in Brazil: A retrospective cohort study.

BACKGROUND: The present study evaluated factors associated with losses in the latent tuberculosis infection (LTBI) cascade of care in contacts of tuberculosis (TB) patients, in a referral center from a highly endemic region in Brazil. METHODS: Contacts of 1672 TB patients were retrospectively studied between 2009 and 2014. Data on TB screening by clinical investigation, radiographic examination and tuberculin skin test (TST) were extracted from medical records. Losses in the cascade of care and TB incidence within 2-year follow-up were calculated. RESULTS: From a total of 1180 TB contacts initially identified, only 495 were examined (58% loss), and 20 were diagnosed with active TB at this stage. Furthermore, 435 persons returned for TST result interpretation and 351 (∼81%) were TST positive. Among those with positive TST, 249 (73%) were treated with isoniazid for 6 months whereas 51 abandoned therapy early. Three individuals who did not receive LTBI treatment, one with incomplete treatment and another who completed treatment developed active TB. A logistic regression analysis revealed that increases in age were associated with losses in the LTBI cascade independent of other clinical and epidemiological characteristics. CONCLUSIONS: Major losses occur at initial stages and older patients are at higher risk of not completing the LTBI cascade of care.

Differential Expression of Activation Markers by Mycobacterium tuberculosis-specific CD4+ T Cell Distinguishes Extrapulmonary From Pulmonary Tuberculosis and Latent Infection.

BACKGROUND: Diagnosis of active tuberculosis (ATB) currently relies on detection of Mycobacterium tuberculosis (Mtb). Identifying patients with extrapulmonary TB (EPTB) remains challenging because microbiological confirmation is often not possible. Highly accurate blood-based tests could improve diagnosis of both EPTB and pulmonary TB (PTB) and timely initiation of anti-TB therapy. METHODS: A case-control study was performed using discriminant analyses to validate an approach using Mtb-specific CD4+T-cell activation markers in blood to discriminate PTB and EPTB from latent TB infection (LTBI) as well as EPTB from PTB in 270 Brazilian individuals. We further tested the effect of human immunodeficiency virus (HIV) coinfection on diagnostic performance. Frequencies of interferon-γ +CD4+T cells expressing CD38, HLADR, and/or Ki67 were assessed by flow cytometry. RESULTS: EPTB and PTB were associated with higher frequencies of CD4+T cells expressing CD38, HLADR, or Ki67 compared with LTBI (all P values < .001). Moreover, frequencies of HLADR+ (P = .03) or Ki67+ (P < .001) cells accurately distinguished EPTB from PTB. HIV infection did not affect the capacity of these markers to distinguish ATB from LTBI or EPTB from PTB. CONCLUSIONS: Cell activation markers in Mtb-specific CD4+T cells distinguished ATB from LTBI and EPTB from PTB, regardless of HIV infection status. These parameters provide an attractive approach for developing blood-based diagnostic tests for both active and latent TB.

Host Inflammatory Biomarkers of Disease Severity in Pediatric Community-Acquired Pneumonia: A Systematic Review and Meta-analysis.

BACKGROUND: Community-acquired pneumonia (CAP) is the leading cause of death in children. Identification of reliable biomarkers offers the potential to develop a severity quantitative score to assist in clinical decision-making and improve outcomes. METHODS: A systematic review and meta-analysis was performed in PubMed and EMBASE on November 13, 2018, to examine the association between host inflammatory biomarkers and CAP severity in children. The inclusion criteria were case-control, cross-sectional, and cohort studies that examined candidate serum biomarkers. We extracted outcomes of interest, means, and standardized mean differences (SMDs) of plasma and serum levels of biomarkers together with information on disease severity. Meta-analysis was performed. This review was registered in the PROSPERO international registry (CRD42019123351). RESULTS: Two hundred seventy-two abstracts were identified, and 17 studies were included. Among the biomarkers evaluated, levels of C-reactive protein (CRP; SMD, 0.63; 95% confidence interval [CI], 0.35 to 0.91), interleukin (IL)-6 (SMD, 0.46; 95% CI, 0.25 to 0.66), IL-8 (SMD, 0.72; 95% CI, 0.15 to 1.29), neutrophil count (SMD, 0.27; 95% CI, 0.07 to 0.47), and procalcitonin (SMD, 0.68; 95% CI, 0.20 to 1.15) were substantially increased in severe CAP. In contrast, IL-2 concentrations (SMD, -0.24; 95% CI, -0.45 to -0.03) were higher in nonsevere CAP. Study heterogeneity was reported to be high (I 2 > 75%), except for IL-2, IL-5, IL-6, and IL-12p70, which were classified as moderate (I 2 = 50%-74%). Only neutrophil and white blood cell counts were described by studies exhibiting a low level of heterogeneity. CONCLUSIONS: Our results suggest that host biomarkers, and especially CRP, IL-6, IL-8, and procalcitonin levels, have the potential to predict severe CAP in pediatric populations.

Hydroxyurea alters circulating monocyte subsets and dampens its inflammatory potential in sickle cell anemia patients.

Sickle cell anemia (SCA) is a hemolytic disease in which vaso-occlusion is an important pathophysiological mechanism. The treatment is based on hydroxyurea (HU), which decreases leukocyte counts and increases fetal hemoglobin synthesis. Different cell types are thought to contribute to vaso-occlusion. Nevertheless, the role of monocytes subsets remains unclear. We investigated frequencies of monocytes subsets in blood and their response to HU therapy, testing their ability to express pro-inflammatory molecules and tissue factor (TF). We identified major changes in monocyte subsets, with classical monocytes (CD14++CD16-) appearing highly frequent in who were not taking HU, whereas those with patrolling phenotype (CD14dimCD16+) were enriched in individuals undergoing therapy. Additionally, HU decreased the production of TNF-α, IL1-ß, IL-6, IL-8 as well as TF by the LPS-activated monocytes. Likewise, frequency of TF-expressing monocytes is increased in patients with previous vaso-occlusion. Moreover, activated monocytes expressing TF produced several pro-inflammatory cytokines simultaneously. Such polyfunctional capacity was dramatically dampened by HU therapy. The frequency of classical monocytes subset was positively correlated with percentage cytokine producing cells upon LPS stimulation. These findings suggest that classical monocytes are the subset responsible for multiple pro-inflammatory cytokine production and possibly drive inflammation and vaso-occlusion in SCA which is damped by HU.

Plasma levels of C-reactive protein, matrix metalloproteinase-7 and lipopolysaccharide-binding protein distinguish active pulmonary or extrapulmonary tuberculosis from uninfected controls in children.

The immune profile associated with distinct clinical forms of tuberculosis (TB) has been extensively described for adult populations. Nevertheless, studies describing immune determinants of pulmonary or extrapulmonary TB (PTB or EPTB, respectively) in children are scarce. Here, we retrospectively assessed plasma levels of several mediators of inflammation in age and sex-matched children from South India presenting with PTB (n = 14) or EPTB (n = 22) as well as uninfected healthy controls (n = 19) to identify biomarkers that could accurately distinguish different TB clinical forms. Furthermore, we performed exploratory analyses testing the influence of sex on the systemic inflammatory profile. The analyses identified a biosignature of 10 biomarkers capable of distinguishing the three clinical groups simultaneously. Machine-learning decision trees indicated that C-reactive protein (CRP), matrix metalloproteinase (MMP)-7 and lipopolysaccharide-binding protein (LBP) were the markers that, when combined, displayed the highest accuracy in identifying the clinical groups. Additional exploratory analyses suggested that the disease signatures were highly influenced by sex. Therefore, sex differentially impacted status of systemic inflammation, immune activation and tissue remodeling in children with distinct clinical forms of TB. Regardless of such nuances related to biological sex, MMP-7, CRP and LBP were strong discriminators of active TB and thus could be considered as biomarkers useful in discrimination different TB clinical forms. These observations have implications on our understanding of the immunopathology of both clinical forms of TB in pediatric patients. If validated by other studies in the future, the combination of identified biomarkers may help development of point-of-care diagnostic or prognostic tools.

Persistent inflammation during anti-tuberculosis treatment with diabetes comorbidity.

Diabetes mellitus (DM) increases risk for pulmonary tuberculosis (TB) and adverse treatment outcomes. Systemic hyper-inflammation is characteristic in people with TB and concurrent DM (TBDM) at baseline, but the impact of TB treatment on this pattern has not been determined. We measured 17 plasma cytokines and growth factors in longitudinal cohorts of Indian and Brazilian pulmonary TB patients with or without DM. Principal component analysis revealed virtually complete separation of TBDM from TB individuals in both cohorts at baseline, with hyper-inflammation in TBDM that continued through treatment completion at six months. By one year after treatment completion, there was substantial convergence of mediator levels between groups within the India cohort. Non-resolving systemic inflammation in TBDM comorbidity could reflect delayed lesion sterilization or non-resolving sterile inflammation. Either mechanism portends unfavorable long-term outcomes including risk for recurrent TB and for damaging immune pathology.

Changes in inflammatory protein and lipid mediator profiles persist after antitubercular treatment of pulmonary and extrapulmonary tuberculosis: A prospective cohort study.

BACKGROUND: The identification of meaningful biomarkers of tuberculosis (TB) has potential to improve diagnosis, disease staging and prediction of treatment outcomes. It has been shown that active pulmonary TB (PTB) is associated with qualitative and quantitative changes in systemic immune profile, suggesting a chronic inflammatory imbalance. Here we characterized the profile of PTB and extrapulmonary TB (EPTB) in a prospective cohort study. METHODS: We measured a panel of 27 inflammatory cytokines, soluble receptors, and lipid mediators in peripheral blood from patients with PTB or EPTB from a prospective clinical study in China. Multidimensional analyses were performed to describe associations between plasma levels of biomarkers and different TB disease presentation profiles. RESULTS: Mycobacterium tuberculosis infection induced changes in both the expression and correlation profiles of plasma mediators of inflammation in patients with PTB compared to those with EPTB. Increases in mycobacterial loads in sputum smears were associated with rises in concentrations of several molecules involved in TB pathogenesis, such as IL-1ß, IFN-α, IL-10 and PGF2α. Moreover, PTB patients presenting with severe disease exhibited a distinct inflammatory profile hallmarked by heightened levels of TNF-α, IL-1ß, IL17, IL-18 and IL-27. Interestingly, while antitubercular treatment (ATT) resulted in early changes of plasma concentrations of markers in PTB, changes were delayed in EPTB patients. Exploratory analyses of the molecular degree of perturbation (MDP) of the inflammatory mediators before and during ATT suggested the occurrence of infection and/or treatment-induced long lasting "inflammatory imprinting" of biomarker profiles in TB. At 24 weeks post ATT commencement, markers underlying the observed increases in MDP scores were IL-27 in PTB and IL-1ß in EPTB patients. CONCLUSION: Our findings describe systemic and durable changes in the concentrations of inflammatory cytokines and lipid mediators in both PTB and EPTB and emphasize the role of M. tuberculosis bacterial burden and site of disease in modulating patient immune biomarkers.

Author Correction: Differential expression of CXCR3 and CCR6 on CD4+ T-lymphocytes with distinct memory phenotypes characterizes tuberculosis-associated immune reconstitution inflammatory syndrome.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Tuberculosis-associated anemia is linked to a distinct inflammatory profile that persists after initiation of antitubercular therapy.

Pulmonary tuberculosis (PTB) is associated with chronic inflammation and anemia. How anemia impacts systemic inflammation in PTB patients undergoing antitubercular therapy (ATT) is not fully understood. In the present study, data on several blood biochemical parameters were retrospectively analyzed from 118 PTB patients during the first 60 days of ATT. Multidimensional statistical analyses were employed to perform detailed inflammatory profiling of patients stratified by anemia status prior to treatment. Anemia was defined as hemoglobin levels <12.5 g/dL for female and <13.5 g/dL for male individuals. The findings revealed that most of anemia cases were likely caused by chronic inflammation. A distinct biosignature related to anemia was detected, defined by increased values of uric acid, C-reactive protein, and erythrocyte sedimentation rate. Importantly, anemic patients sustained increased levels of several biochemical markers at day 60 of therapy. Preliminary analysis failed to demonstrate association between persistent inflammation during ATT with frequency of positive sputum cultures at day 60. Thus, TB patients with anemia exhibit a distinct inflammatory profile, which is only partially reverted at day 60 of ATT.

Differential expression of CXCR3 and CCR6 on CD4+ T-lymphocytes with distinct memory phenotypes characterizes tuberculosis-associated immune reconstitution inflammatory syndrome.

Immune reconstitution inflammatory syndrome (IRIS) occurs in up to 40% of individuals co-infected with pulmonary tuberculosis (PTB) and HIV, primarily upon antiretroviral therapy (ART) initiation. Phenotypic changes in T-cells during TB-IRIS and their relationship with systemic inflammation are not fully understood. In this prospective cohort study, we followed 48 HIV-positive patients with PTB from South India before and after ART initiation, examining T-lymphocyte subsets and inflammatory biomarkers in peripheral blood. Quantification of naïve (CD27+CD45RO-) as well as effector memory CD4+ T cells (CD27-CD45RO+) at weeks 2-6 after ART initiation could distinguish TB-IRIS from non-IRIS individuals. Additional analyses revealed that ART reconstituted different quantities of CD4+ T lymphocyte subsets with preferential expansion of CXCR3+ CCR6- cells in TB-IRIS patients. Moreover, there was an expansion and functional restoration of central memory (CD27+CD45RO+) CXCR3+CCR6- CD4+ lymphocytes and corresponding cytokines, with reduction in CXCR3-CCR6+ cells after ART initiation only in those who developed TB-IRIS. Together, these observations trace a detailed picture of CD4+ T cell subsets tightly associated with IRIS, which may serve as targets for prophylactic and/or therapeutic interventions in the future.

Generation and Characterization of a Dual-Reporter Transgenic Leishmania braziliensis Line Expressing eGFP and Luciferase.

In this study, we generated a transgenic strain of Leishmania braziliensis, an etiological agent associated with a diversity of clinical manifestations of leishmaniasis ranging from localized cutaneous to mucocutaneous to disseminated disease. Transgenic parasites expressing reporter proteins are valuable tools for studies of parasite biology, host-pathogen interactions, and anti-parasitic drug development. To this end, we constructed an L. braziliensis line stably expressing the reporters eGFP and luciferase (eGFP-LUC L. braziliensis). The integration cassette co-expressing the two reporters was targeted to the ribosomal locus (SSU) of the parasite genome. Transgenic parasites were characterized for their infectivity and stability both in vitro and in vivo. Parasite maintenance in axenic long-term culture in the absence of selective drugs did not alter expression of the two reporters or infection of BALB/c mice, indicating stability of the integrated cassette. Infectivity of eGFP-LUC, L. braziliensis, both in vivo and in vitro was similar to that obtained with the parental wild type strain. The possibility of L. braziliensis tracking and quantification using fluorescence and luminescence broadens the scope of research involving this neglected species, despite its importance in terms of public health concerning the leishmaniasis burden.

Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites.

Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24-48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections.