Discovery and Validation of a Three-Cytokine Plasma Signature as a Biomarker for Diagnosis of Pediatric Tuberculosis.

Pediatric TB poses challenge in diagnosis due to the paucibacillary nature of the disease. We conducted a prospective diagnostic study to identify immune biomarkers of pediatric TB and controls (discovery cohort) and obtained a separate "validation" cohort of confirmed cases of pediatric TB and controls. Multiplex ELISA was performed to examine the plasma levels of cytokines. Discovery and validation cohorts revealed that baseline plasma levels of IFNγ, TNFα, IL-2, and IL-17A were significantly higher in active TB (confirmed TB and unconfirmed TB) in comparison to unlikely TB children. Receiver operating characteristics (ROC) curve analysis revealed that IFNγ, IL-2, TNFα, and IL-17A (in the discovery cohort) and TNFα and IL-17A (in the validation cohort) could act as biomarkers distinguishing confirmed or unconfirmed TB from unlikely TB with the sensitivity and specificity of more than 90%. In the discovery cohort, cytokines levels were significantly diminished following anti-tuberculosis treatment. In both the cohorts, combiROC models offered 100% sensitivity and 98% to 100% specificity for a three-cytokine signature of TNFα, IL-2, and IL-17A, which can distinguish confirmed or unconfirmed TB children from unlikely TB. Thus, a baseline cytokine signature of TNFα, IL-2, and IL-17A could serve as an accurate biomarker for the diagnosis of pediatric tuberculosis.

Deciphering the Role of Mucosal Immune Responses and the Cervicovaginal Microbiome in Resistance to HIV Infection in HIV-Exposed Seronegative (HESN) Women.

The female genital tract (FGT) is an important site of human immunodeficiency virus (HIV) infection. Discerning the nature of HIV-specific local immune responses is crucial for identifying correlates of protection in HIV-exposed seronegative (HESN) individuals. The present study involved a comprehensive analysis of soluble immune mediators, secretory immunoglobulins (sIg), natural killer (NK) cells, CXCR5+ CD8+ T cells, T follicular helper (Tfh) cells, and T regulatory cells (Tregs) in the vaginal mucosa as well as the nature and composition of the cervicovaginal microbiome in HESN women. We found significantly elevated antiviral cytokines, soluble immunoglobulins, and increased frequencies of activated NK cells, CXCR5+ CD8+ T cells, and Tfh cells in HESN females compared to HIV-unexposed healthy (UH) women. Analysis of the genital microbiome of HESN women revealed a greater bacterial diversity and increased abundance of Gardnerella spp. in the mucosa. The findings suggest that the female genital tract of HESN females represents a microenvironment equipped with innate immune factors, antiviral mediators, and critical T cell subsets that protect against HIV infection. IMPORTANCE The vast majority of human immunodeficiency virus (HIV) infections across the world occur via the sexual route. The genital tract mucosa is thus the primary site of HIV replication, and discerning the nature of HIV-specific immune responses in this compartment is crucial. The role of the innate immune system at the mucosal level in exposed seronegative individuals and other HIV controllers remains largely unexplored. This understanding can provide valuable insights to improve vaccine design. We investigated mucosal T follicular helper (Tfh) cells, CXCR5+ CD8+ T cells, natural killer (NK) cells subsets, soluble immune markers, and microbiome diversity in HIV-exposed seronegative (HESN) women. We found a significantly higher level of mucosal CXCR5+ CD8+ T cells, CD4+ Tfh cells, activated NK cell subsets, and antiviral immune cell mediators in HESN women. We also found a higher abundance of Gardnerella spp., microbiome dysbiosis, and decreased levels of inflammatory markers to be associated with reduced susceptibility to HIV infection. Our findings indicate that increased distribution of mucosal NK cells, CXCR5+ CD8+ T cells, Tfh cells, and soluble markers in HIV controllers with a highly diverse cervicovaginal microbiome could contribute effectively to protection against HIV infection. Overall, our findings imply that future vaccine design should emphasize inducing these highly functional cell types at the mucosal sites.

Elevated Numbers of HIV-Specific Poly-Functional CD8+ T Cells With Stem Cell-Like and Follicular Homing Phenotypes in HIV-Exposed Seronegative Individuals.

HIV-specific CD8+ T cells are known to play a key role in viral control during acute and chronic HIV infection. Although many studies have demonstrated the importance of HIV-specific CD8+ T cells in viral control, its correlation with protection against HIV infection remains incompletely understood. To better understand the nature of the immune response that contributes to the early control of HIV infection, we analyzed the phenotype, distribution and function of anti-viral CD8+ T cells in a cohort of HIV-exposed seronegative (HESN) women, and compared them with healthy controls and HIV-infected individuals. Further, we evaluated the in vitro viral inhibition activity of CD8+ T cells against diverse HIV-1 strains. We found that the HESN group had significantly higher levels of CD8+ T cells that express T-stem cell-like (TSCM) and follicular homing (CXCR5+) phenotype with more effector like characteristics as compared to healthy controls. Further, we observed that the HESN population had a higher frequency of HIV-specific poly-functional CD8+ T cells with robust in vitro virus inhibiting capacity against different clades of HIV. Overall, our results demonstrate that the HESN population has elevated levels of HIV-specific poly-functional CD8+ T cells with robust virus inhibiting ability and express elevated levels of markers pertaining to TSCM and follicular homing phenotype. These results demonstrate that future vaccine and therapeutic strategies should focus on eliciting these critical CD8+ T cell subsets.

Evaluation of antiviral T cell responses and TSCM cells in volunteers enrolled in a phase I HIV-1 subtype C prophylactic vaccine trial in India.

T cells play an important role in controlling viral replication during HIV infection. An effective vaccine should, therefore, lead to the induction of a strong and early viral-specific CD8+ T cell response. While polyfunctional T cell responses are thought to be important contributors to the antiviral response, there is evidence to show that polyfunctional HIV- specific CD8+ T cells are just a small fraction of the total HIV-specific CD8+ T cells and may be absent in many individuals who control HIV replication, suggesting that other HIV-1 specific CD8+ effector T cell subsets may be key players in HIV control. Stem cell-like memory T cells (TSCM) are a subset of T cells with a long half-life and self-renewal capacity. They serve as key reservoirs for HIV and contribute a significant barrier to HIV eradication. The present study evaluated vaccine-induced antiviral responses and TSCM cells in volunteers vaccinated with a subtype C prophylactic HIV-1 vaccine candidate administered in a prime-boost regimen. We found that ADVAX DNA prime followed by MVA boost induced significantly more peripheral CD8+ TSCM cells and higher levels of CD8+ T cell-mediated inhibition of replication of different HIV-1 clades as compared to MVA alone and placebo. These findings are novel and provide encouraging evidence to demonstrate the induction of TSCM and cytotoxic immune responses by a subtype C HIV-1 prophylactic vaccine administered using a prime-boost strategy.

Pediatric presentations of leptospirosis.

OBJECTIVE: Leptospirosis in children is an often under diagnosed condition due to the non specificity of the presentations except for the classical Weil's disease. METHODS: Children presenting with symptoms and signs suggestive of Leptospirosis were included in the study. Diagnostic criteria were fever, myalgia, conjunctival suffusion, Jaundice, headache, altered sensorium, seizures, bleeding manifestation and oliguria. Their clinical profile, lab parameters (general and specific), response to treatment and outcome were analysed. RESULT: One hundred and thirty nine cases were diagnosed during a 4-year period. The commonest symptoms were fever 133 (96%), headache and myalgia 34 (24%). Jaundice was present in only 25 (18%) of cases with renal failure in 2 cases. The frequently encountered clinical signs were hepatomegaly in 100 (72%), myalgia in 34 (24%) with icterus in 25 (18%), 12 (9%) of children presented with shock and 10 (7%) had meningitis. CPK estimated was a useful index of myositis. The diagnosis was confirmed by Dark field microscopy and paired or single high serological tests (MAT, ELISA IgM). Overlapping infections such as culture positive Salmonella typhi with leptospirosis (Serology positive) or Dengue Hemorrhagic fever with Leptospirosis presented with complications such as a myocarditis, shock and ARDS. CONCLUSION: Presentation of non-icteric forms of Leptospirosis are often non-specific and may be missed unless there is a high index of suspicion. This study emphasizes the myositis and meningitis forms of leptospirosis. Delayed diagnosis leads to increased mortality and morbidity.

Importance of screening the peripheral smear.

A 5-year-old boy presented with history of failure to thrive from infancy. There was a history of one sibling death due to similar problems and history of severe abortions in the mother. Routine examination of peripheral smear revealed more than 50% acanthocytes. Based on this tests were streamlined to doing lipid profile and Lipo protein electrophoresis which revealed hypolipidemia and absent beta hypo protein band. Jejunal mucosal biopsy confirmed the diagnosis of A Beta Lipo proteinemia which revealed lipid laden enterocytes. This case illustrates the importance of simple tests like peripheral smear examination in streamlining further tests in the diagnosis of major diseases.

Circulating biomarkers of pulmonary and extrapulmonary tuberculosis in children.

Tuberculosis (TB) in children is not only more likely to cause more severe disease than that seen in adults, it is also more likely to be extrapulmonary. Moreover, pediatric TB is very difficult to diagnose and suffers from a lack of understanding of host biomarkers for monitoring the progression of disease. Hence, we sought to identify the expression patterns of a variety of biomarkers in the plasma of children with pulmonary TB (PTB) and extrapulmonary TB (ETB), as well as in healthy control (HC) children. Thus, we examined a variety of circulating markers reflecting tissue inflammation, oxidative stress, innate immune activation, fibrosis, and the cytokine response. Children with active TB, compared to HC children, showed markedly elevated plasma levels of matrix metalloproteinases and their endogenous inhibitors. In addition, children with active TB had significantly elevated levels of C-reactive protein, α-2 macroglobulin, and haptoglobin, as well as hemoxygenase 1. Markers of innate immune activation (lipopolysaccharide [LPS] and lipopolysaccharide-binding protein [LBP]) were significantly lower in ETB than in PTB children. Although there were no significant differences between the two groups in their levels of cytokines (type 1 [gamma interferon (IFN-γ), tumor necrosis factor α (TNF-α), interleukin 2 (IL-2), and IL-12], type 2 [IL-4, IL-5, IL-13, and IL-33], and most type 17 [IL-17A, IL-22, IL-1ß, and IL-6] and type 1 interferons [IFN-α and IFN-ß]) or most of the cytokines associated with immune modulation (IL-10 and IL-20), pediatric TB was associated with elevated plasma transforming growth factor ß (TGF-ß), IL-21, and IL-23 levels. Thus, pediatric TB is characterized by elevated levels of a variety of biomarkers at homeostasis, suggesting that these responses may play a crucial role in disease pathogenesis.

The curious case of a button which led to the needle.

Foreign bodies in the heart are uncommon in children. These are often removed even if asymptomatic to prevent complications like erosion, embolization, bleeding, thrombosis, and endocarditis. We report the case of a one-and-a-half-year-old child with a hypodermic needle in the heart which was found incidentally and removed successfully by surgery.

Suppressed type 1, type 2, and type 17 cytokine responses in active tuberculosis in children.

Type 1 cytokine responses are known to play an important role in immunity to tuberculosis (TB) in children, although little is known about other factors that might be important. In addition, children are more prone to developing extrapulmonary manifestations of TB than adults. To identify the immune responses important both in control of infection and in extrapulmonary dissemination, we examined mycobacterium-specific cytokine responses of children with pulmonary TB (PTB) and extrapulmonary TB (ETB) and compared them with those of healthy control children (HC). No significant differences were found in the cytokine responses either with no stimulation or following mycobacterial-antigen (Ag) stimulation between children with PTB and ETB. On the other hand, children with active TB compared with HC showed markedly diminished production of type 1 (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), 2 (interleukin 4 [IL-4] and IL-13), and 17 (IL-17A, IL-21, and IL-23)-associated cytokines with no stimulation and in response to mycobacterial antigens. This was not associated with significantly altered production of IL-10 or transforming growth factor ß (TGF-ß). Among children with ETB, those with neurologic involvement exhibited more significantly diminished Ag-driven IFN-γ and IL-17 production. Pediatric TB is characterized by diminished type 1, 2, and 17 cytokine responses, with the most profound diminution favoring development of neurologic TB, suggesting a crucial role for these cytokines in protection against pediatric tuberculosis.

GAP between knowledge and skills for the implementation of the ACCM/PALS septic shock guidelines in India: is the bridge too far?

OBJECTIVE: To determine whether physicians were aware of and had the skills to implement the American College of Critical Care Medicine/Pediatric Advanced Life Support Course septic shock protocol. DESIGN: A cross-sectional questionnaire survey. SETTING: Four academic institutions in Chennai, Manipal, Mangalore, and Trivandrum - cities representing the three southern states of Tamil Nadu, Karnataka, and Kerala, respectively, between February and April 2006. INTERVENTIONS: Pre and post lecture questions. They were evaluated using 11 questions testing knowledge and 10 questions testing their comfort level in performing interventions related to the initial resuscitation in septic shock. MEASUREMENT AND MAIN RESULT: The ACCM/PALS sepsis guidelines were taught during the PALS course conducted in the four academic institutions. A total of 118 delegates participated, of whom 114 (97%) were pediatricians and four (3%) were anaesthetists. The overall mean number of correct responses for the 11 questions testing knowledge before and after the lecture was 2.1 and 4.07, respectively P=0.001(paired t test). Although, 42% of the respondents (n=50) were aware of the ACCM guidelines, 88% (n=104) did not adhere to it in their practice. A total of 86% (n=101) and 66% (n=78) did not feel comfortable titrating inotropes or intubating in the ED; 78% (n=92) and 67% (n=78), respectively felt that central venous access (CVA) and arterial pressure (AP) monitoring were unimportant in the management of fluid refractory shock. Of the physicians, 20% (n=24) had never intubated a patient, 78% (n=92) had not introduced a central venous catheter, and 76% (n=90) had never introduced an intra-arterial catheter. CONCLUSIONS: In view of the lack of skills and suboptimal knowledge, the ACCM/PALS sepsis guidelines may be inappropriate in its current format in the Indian setting. More emphasis needs to be placed on educating community pediatricians with a simpler clinical protocol, which has the potential to save many more children.