Ação antifúngica da lactoferrina frente Candida non-albicans isoladas da cavidade oral de crianças infectadas pelo HIV / Antifungal action of lactoferrin against Candida non-albicans isolated from the oral cavity of HIV-infected children

A lactoferrina é uma glicoproteína de ligação ao ferro, que está presente na saliva, no leite, e em outras secreções exócrinas. Essa proteína tem inúmeras funções biológicas, incluindo efeitos antifúngicos e antibacterianos, além de imunomoduladores. O objetivo do presente estudo foi avaliar, in vitro, pela primeira vez, a atividade antifúngica da lactoferrina contra cepas de Candida nãoalbicans isoladas da cavidade oral de crianças infectadas pelo HIV e crianças saudáveis. Além disso, mensurar a degradação da lactoferrina por essas cepas através de SDS-PAGE (análise eletroforética em gel de poliacrilamida) e determinar, in vitro, a concentração mínima inibitória de lactoferrina capaz de matar 50% das células de Candida não-albicans. Cepas de Candida spp. foram obtidas através da coleta de saliva de 70 crianças infectadas pelo HIV e 50 crianças sem evidências de imunossupressão, com idade de 3 a 13 anos (ALVES, 2014). As diferentes espécies de Candida foram identificadas através de assimilação e fermentação de açúcar (API 20C, Biomérieux, França). Para o ensaio, in vitro, de morte de celular na presença de lactoferrina, 24 isolados de Candida, entre elas parapsilosis, tropicalis, krusei, guillermondi e dubliniensis, foram selecionados. Para a realização da análise por SDS-PAGE, cepas de todas as espécies consideradas resistentes nos ensaios de morte celular, foram incluídas. O porcentual de morte celular (%) de Candida não- albicans com a adição de 100 µg/ml de lactoferrina variou de 3,1% (C. dubliniensis) a 88,1% (C. tropicalis) no grupo HIV, e de 14,1% a 30,37% no grupo não-HIV (C . parapsilosis). Não houve correlação entre a densidade celular e o percentual de morte celular. C. dubliniensis foi a espécie mais resistente a lactoferrina e o porcentual de morte celular de C. parapsilosis foi significativamente maior em comparação com C. krusei na concentração de 1X104 células/ml (p = 0,033). Todos os isolados foram capazes de degradar a lactoferrina na concentração 1x108 células/ml, especialmente C. parapsilosis, pelo grupo HIV. Além disso, a lactoferrina na concentração de 500 µg/ml foi capaz de matar mais de 50% das células apenas dos isolados de C. tropicalis e C. guilliermondii. Concluiu-se que a lactoferrina tem atividade antifúngica contra Candida não-albicans de crianças infectadas pelo HIV, mas algumas espécies apresentam alguma resistência a esta proteína. Além disso, todas as Candida spp. foram capazes de degradar a lactoferrina quando estavam em concentração de 1X108 cels/ml, e a lactoferrina na concentração de 500µl/ml foi capaz de matar mais que 50% das células de C. tropicalis e C. guillermondii

Lactoferrin is an iron-binding glycoprotein, which is present in saliva, milk and other exocrine secretions. This protein has a number of biological functions, including antifungical, antimicrobial and immunomodulatory effects. The aim of this study was to evaluate, for the first time, the antifungal activity of lactoferrin against isolates of Candida spp. from the oral cavity of HIV-infected children and children with no clinical evidence of immunosuppression. Furthermore, measure, in vitro, the degradation of lactoferrin by Candida spp. through SDS-PAGE (electrophoretic analysis on polyacrylamide gel) and determine, in vitro, the minimum inhibitory concentration of lactoferrin able to kill 50% of cells of Candida non-albicans. Strains of Candida spp. were obtained through saliva collect of 70 HIV-infected children and 50 children without evidence of immunosuppression ageing between 3 to13 years old (ALVES, 2014). All Candida species were identified by sugar assimilation and fermentation (API 20C, Biomerieux, France). For the in vitro study, death of lactoferrin of 24 Candida isolates, including parapsilosis, tropicalis, krusei, guillermondii, and dubliniensis, were selected. To perform the analysis by SDS-PAGE, strains of all available species considered resistants, which were observed at lactoferrin death`s study, were included. The cell death percentage (%) of non-albicans Candida by addition of 100 µg of lactoferrin range from 3.1% (C. dublinienes) to 88.1% (C. tropicalis) in HIV group, and 14.1% to 30.37% in N-HIV (C. parapsilosis), but there was no correlation between cell density and death %. C. dubliniensis was the most resistant specie to lactoferrin and the cell death % of C. parapsilosis was significant higher comparing to C. krusei at 1X104 cells/ml (p=0.033). All the isolates were able to degrade lactoferrin at 108 cells/ml, mainly C. parapsilosis from HIV. Furthermore, lactoferrin at 500 µg/ml concentration was able to kill more than 50% of the cells only for C. tropicalis and C. guillermondii isolates. It was concluded that lactoferrin has antifungal activity against non-albicans Candida from HIV children, but some species present some resistance to this protein. Furthermore, all Candida spp. were able to degrade lactoferrin when was at concentration of 1x108 cells / ml, also, lactoferrin at concentration 500µl / ml was able to kill more than 50% of C. tropicalis and C. guillermondii cells.

The "moral career" of perinatally HIV-infected children: revisiting Goffman's concept.

HIV-infected children usually live in vulnerable situations, experiencing discrimination and stigma commonly felt by other people living with HIV/AIDS. The present study aims to analyse primary socialisation of HIV-infected children and adolescents recruited from a public health service in Rio de Janeiro (Brazil) as a social process that shapes a new generation of stigmatised and vulnerable persons. Research was informed by an interactionist perspective, focusing on key aspects of HIV-infected children and adolescents life histories under the conceptual frame of Erving Goffman's theories regarding "moral careers". Goffman defines the making of a moral career as the process through which a person learns that she/he possesses a particular attribute, which may lead her/him to be discredited by members of the surrounding society. We have identified aspects of life histories of HIV-vertically infected children and adolescents for each aspect of "moral career" as described by Goffman, relating them to as family structure, the experience of living HIV within the family, and the position and family role of a given subject. The patterns of "moral career" proposed by Goffman in 1963 were useful in identifying components of HIV-related stigma among children and adolescents. These include gender and social disadvantages, difficulty in coping with a child with a potentially severe disease, orphanhood, abandonment, adoption and disclosure of one's HIV serostatus. Primary socialisation of HIV-infected children and adolescents is a key piece of the complex HIV/AIDS-labelling process that could be targeted by interventions aiming to decrease stigma and marginalisation. Health care workers and stakeholders should be committed to ensuring education and guaranteeing the legal rights of this specific population, including the continuous provision of quality health care, full access to school and support to full disclosure of HIV diagnosis.

Viral suppression and adherence among HIV-infected children and adolescents on antiretroviral therapy: results of a multicenter study / Supressão viral e adesão entre crianças e adolescentes vivendo com HIV na terapia antirretroviral: resultados de um estudo multicêntrico

OBJECTIVE: To evaluate treatment adherence among perinatally-infected pediatric human immunodeficiency virus (HIV) patients followed in pediatric centers in Brazil. METHODS: This was a cross-sectional multicenter study. Medical records were reviewed and adherence scale, assessment of caregivers' quality of life (WHOQOL-BREF), anxiety, depression, and alcohol/substances use/abuse were assessed. Outcomes included self-reported 100% adherence in the last three days and HIV viral load (VL) < 50 copies/mL. Statistical analyses included contingency tables and respective statistics, and multivariable logistic regression. RESULTS: 260 subjects were enrolled: 78% children and 22% adolescents; 93% of caregivers for the children and 77% of adolescents reported 100% adherence; 57% of children and 49% of adolescents had VL < 50 copies/mL. In the univariate analyses, HIV diagnosis for screening due to maternal infection, lower caregiver scores for anxiety, and higher scores in physical and psychological domains of WHOQOL-BREF were associated with 100% adherence. Shorter intervals between pharmacy visits were associated with VL < 50 copies/mL (p ≤ 0.01). Multivariable regression demonstrated that caregivers who did not abuse alcohol/other drugs (OR = 0.49; 95% CI: 0.27-0.89) and median interval between pharmacy visits < 33 days (OR = 0.97; 95% CI: 0.95-0.98) were independently associated with VL < 50 copies/mL; whereas lower caregiver scores for anxiety (OR = 2.57; 95% CI: 1.27-5.19) and children's HIV diagnosis for screening due to maternal infection (OR = 2.25; 95% CI: 1.12-4.50) were found to be independently associated with 100% adherence. CONCLUSIONS: Pediatric HIV programs should perform routine assessment of caregivers' quality of life, and anxiety and depression symptoms. In this setting, pharmacy records are essential to help identify less-than-optimal adherence. .

OBJETIVO: Avaliar a adesão ao tratamento antirretroviral entre portadores de HIV acompanhados em centros pediátricos. MÉTODOS: Trata-se de estudo transversal multicêntrico. Os prontuários ambulatoriais foram revistos e aplicadas escala de adesão, avaliação de qualidade de vida (WHOQOL-BREF), ansiedade, depressão e uso indevido de álcool/substâncias entre cuidadores. Os desfechos incluíram autorrelato 100% de adesão nos últimos três dias e carga viral do HIV (CV) < 50 cópias/mL. RESULTADOS: 260 indivíduos foram incluídos, 79% crianças e 21% adolescentes; 93% das crianças e 77% dos adolescentes relataram 100% de adesão; 57% das crianças e 49% dos adolescentes tinham CV < 50 cópias /mL. Na análise univariada, diagnóstico do HIV por triagem devido à infecção materna, cuidador com pontuação menor para ansiedade e maior nos domínios físico e psicológico do WHOQOL-BREF se mostraram independentemente associados a 100% de adesão. Intervalos mais curtos entre visitas de farmácia foram associados com CV < 50 cópias /mL (p ≤ 0,01). Regressão multivariada mostrou que os cuidadores sem abuso de álcool/outras drogas (OR = 0,49; IC95% 0,27-0,89) e o intervalo médio entre visitas de farmácia < 33 dias (OR = 0,97; IC95% 0,95-0,98) foram associados com CV < 50 cópias/mL; cuidador com menores escores para ansiedade (OR = 2,57; IC95% 1,27-5,19) e diagnóstico de crianças por triagem devido à infecção materna (OR = 2,25; IC95% 1,12-4,50) foram associados com 100% de adesão. CONCLUSÕES: Programas de HIV pediátrico devem avaliar qualidade de vida e sintomas de ansiedade e depressão dos cuidadores. Registros de farmácia são essenciais na identificação de adesão ...

Viral suppression and adherence among HIV-infected children and adolescents on antiretroviral therapy: results of a multicenter study.

OBJECTIVE: To evaluate treatment adherence among perinatally-infected pediatric human immunodeficiency virus (HIV) patients followed in pediatric centers in Brazil. METHODS: This was a cross-sectional multicenter study. Medical records were reviewed and adherence scale, assessment of caregivers' quality of life (WHOQOL-BREF), anxiety, depression, and alcohol/substances use/abuse were assessed. Outcomes included self-reported 100% adherence in the last three days and HIV viral load (VL)<50 copies/mL. Statistical analyses included contingency tables and respective statistics, and multivariable logistic regression. RESULTS: 260 subjects were enrolled: 78% children and 22% adolescents; 93% of caregivers for the children and 77% of adolescents reported 100% adherence; 57% of children and 49% of adolescents had VL<50 copies/mL. In the univariate analyses, HIV diagnosis for screening due to maternal infection, lower caregiver scores for anxiety, and higher scores in physical and psychological domains of WHOQOL-BREF were associated with 100% adherence. Shorter intervals between pharmacy visits were associated with VL<50 copies/mL (p ≤ 0.01). Multivariable regression demonstrated that caregivers who did not abuse alcohol/other drugs (OR=0.49; 95% CI: 0.27-0.89) and median interval between pharmacy visits<33 days (OR=0.97; 95% CI: 0.95-0.98) were independently associated with VL<50 copies/mL; whereas lower caregiver scores for anxiety (OR=2.57; 95% CI: 1.27-5.19) and children's HIV diagnosis for screening due to maternal infection (OR=2.25; 95% CI: 1.12-4.50) were found to be independently associated with 100% adherence. CONCLUSIONS: Pediatric HIV programs should perform routine assessment of caregivers' quality of life, and anxiety and depression symptoms. In this setting, pharmacy records are essential to help identify less-than-optimal adherence.

Salivary lactoferrin in HIV-infected children: correlation with Candida albicans carriage, oral manifestations, HIV infection and its antifungal activity.

OBJECTIVES: This study aimed to evaluate the concentration of lactoferrin in the saliva of HIV infected and healthy children and analyze the associations between lactoferrin levels, Candida sp. colonization, oral manifestations and medical data. Also, the antifungal ability of lactoferrin to inhibit the growth of Candida albicans isolated from saliva of these children was investigated in vitro. SUBJECTS AND METHODS: Saliva was collected from 70 HIV-infected and 50 healthy children, followed by oral manifestation assessments. The salivary lactoferrin was quantified using an ELISA Kit. The salivary samples were cultured and the Candida spp. colonies counted and then identified by sugar assimilation and fermentation. The antifungal activity of lactoferrin was analyzed in vitro with 10 isolates of C. albicans from each group. RESULTS: The HIV infected children (mean age 9.8 ± 2.8) had higher lactoferrin levels (median 6.13 µg/ml (3.58-7.89)) and were colonized three times more by Candida sp. than the control group (mean age 9.4 ± 2.4) (median 5.74 µg/ml (3.12-6.86)) (p=0.003). Statistical associations were found considering the salivary lactoferrin levels and Candida sp. and oral manifestations between the groups. No associations between lactoferrin concentrations and oral manifestations, immunosuppression, presence of AIDS and use of HAART were observed in the HIV group. The percentage of dead C. albicans due to lactoferrin was inversely proportional to C. albicans cell density for both groups (p<0.001). CONCLUSIONS: HIV-infected children have higher concentrations of lactoferrin and it was associated with Candida sp. colonization but no association with medical data was found. Also, both groups showed similar lactoferrin antifungal activity.

Potencial cariogênico de Candida não-albicans isoladas do biofilme dentário de crianças infectadas pelo HIV / Cariogenic potential of Candida non-albicans isolated from the dental biofilm of HIV-infected children

O objetivo deste estudo foi analisar, in vitro, o padrão de desmineralização do esmalte bovino exposto a C. não-albicans isoladas de biofilme dental de crianças infectadas pelo HIV, em comparação com C. albicans (n = 2) e Streptococcus (S) mutans (ATCC). Também foram avaliadas as formações de biofilme e produção de L-lactato por estas espécies não-albicans. Cento e oitenta blocos de esmalte foram divididos em 6 grupos com biofilme formado por: C. glabrata; C. parapsilosis; C. tropicalis; C. albicans; S. mutans e controle negativo (meio sem biofilme). Três blocos de esmalte de cada grupo foram retirados nos dias 3, 5, 8, 15 e 28 após a formação de biofilme para avaliar a microdureza superficial do esmalte final (SMH). Além disso, a viabilidade das células do biofilme e o L-lactato produzidos por estas células foi medida por redução de XTT e enzimaticamente, respectivamente. Análise microscópica de varrimento laser confocal (CLS) foi utilizado para avaliar a topografia de cada isolado de biofilme de C. não-albicans. Os resultados da análise de SMH foram submetidos a testes de Mann-Whitney de Kruskall-Wallis e com um nível de significância de 95% (p <0,05), enquanto que os dados de viabilidade do biofilme, L-lactato e análise CLS foram avaliadas de forma descritiva. Todos os grupos mostraram um aumento na perda de SMH, mas apenas os grupos expostos a biofilmes de S. mutans e C. albicans foram estatisticamente significativas ao longo do tempo. Ao comparar as espécies C. não-albicans entre si, C. tropicalis apresentaram maior perda% no 5 º dia em comparação com C. parapsilosis e no 8 º dia em comparação com C. glabrata. Não foi observada diferença estatística entre C. parapsilosis e C. glabrata. Todos os isolados de C. não-albicans mostraram o mesmo perfil de formação de biofilme. A produção de L-lactato foi maior em C. tropicalis seguido por C. glabrata e C. parapsilosis. No entanto, a análise mostrou CLS C. parapsilosis biofilme com mais células viáveis e com a matriz extracelular mais. Embora com menor intensidade em comparação com C. albicans e S. mutans, os isolados de Candida não-albicans do biofilme dental de crianças infectadas pelo HIV, foram capazes de causar a desmineralização do esmalte bovino. (AU)

This study aimed to analyzed , in vitro, the demineralization pattern of bovine enamel exposed to biofilm of C. non-albicans isolated from dental biofilm of HIV infected children, comparing to C. albicans (n=2) and Streptococcus (S) mutans (ATCC).We also evaluated the biofilm formations and the production of L-lactate by these nonalbicans species. A hundred eigthy enamel blocks randomly assigned to 6 groups with biofilm formed by: C. glabrata; C. parapsilosis; C. tropicalis; C. albicans; S. mutans and negative control (medium without biofilm). Three enamel blocks from each group were removed on days 3, 5, 8, 15 and 28 after biofilm formation to evaluate the final enamel surface microhardness (SMH). In addition, the cells viability of the biofilm and the L-lactate produced by these cells were measured by XTT reduction and enzymatically, respectively. Confocal laser scanning microscopic (CLS) analysis was used to evaluate the biofilm architecture of each C. non-albicans isolate. The results from SMH analysis were subjected to Kruskall-Wallis and MannWhitney tests with a 95% significance level (p<0.05), while the data from biofilm viability, L-lactate and CLS analysis were evaluated descriptively. All groups showed an increase in loss of SMH but only the groups exposed to biofilms from S. mutans and C. albicans were statistically significant over time. When comparing the species C. non-albicans among themselves, C. tropicalis showed a greater % loss on the 5th day compared with C. parapsilosis and at the 8th day compared to c. glabrata. No statistical difference betwwen C. parapsilosis and C. glabrata was observed. All isolates from C. non-albicans showed the same profile of biofilm formation. The Llactate production was higher in C. tropicalis followed by C. glabrata and C. parapsilosis. However, the CLS analysis showed C. parapsilosis biofilm with more viable cells and with more extracellular matrix. Although with less intensity comparing to C. albicans and S. mutans, isolates of Candida non-albicans from HIV infected children dental biofilm, were able to cause demineralization of bovine enamel. (AU)

Circadian cardiac autonomic function in perinatally HIV-infected preschool children

The 24-h heart rate variability and QT-interval adaptation was investigated in perinatally HIV-infected preschool children classified according to immunological status in order to assess autonomic function at early stages of infection. Thirty-five perinatally HIV-infected and clinically stable children (4.8 ± 0.3 years) were enrolled after approval of the study by the University Hospital Pedro Ernesto Ethics Committee and written informed parental consent was obtained. The children were classified according to peripheral CD4+ count (cells/µL) as follows: group 1, N = 11 (≥1000); group 2, N = 7 (≥500 and <1000); group 3, N = 17 (<500). Left ventricular ejection fraction (>55%), 24-h RR interval variability (RRV) indexes (NN, SDANN, SDNN index, r-MSSD) and 24-h QT and Bazett-corrected QT (QTc) were determined, and groups were matched for age, body surface area, and left ventricular ejection fraction, reducing biases in RRV. The peak differences (∆) between the highest and lowest RRV and QT indexes were extracted from nocturnal (1 am-6 am) and daytime (1 pm-6 pm) hourly assessed segments, respectively. Pearson’s correlation (r) and Kruskal-Wallis ANOVA were used to compare groups. CD4+ count correlated positively with ∆NN (r = 0.45; P = 0.003). There were no significant differences in daytime NN among groups. Nighttime SDNN index (P = 0.01), nighttime r-MSSD (P = 0.003), ∆NN (P = 0.01), ∆SDNN index (P = 0.03) and ∆r-MSSD (P = 0.004) were significantly lower in group 3 than in the other groups. Expected nighttime QTc-interval lengthening was not observed in all groups. In perinatally HIV-infected preschool children with preserved left ventricular systolic function, parasympathetic-mediated autonomic dysfunction parallels immune status, impairing both RRV and circadian QTc interval adaptation.

HIV-1: avaliação da resistência às drogas antiretrovirais em pacientes pediátricos / HIV-1: evaluation of resistance to antiretroviral drugs in pediatric patients

A utilização da terapia antiretroviral, atualmente mais amplamente acessível, implica na permanência da identificação da resistência viral e monitoramento da doença como itens importantes em adultos e pacientes pediátricos infectados pelo vírus da imunodeficiência humana tipo 1. Os principais marcadores da infecção por HIV-1, utilizados no monitoramento da infecção e curso da doença, são as contagens de células T CD4+ e a carga viral. Ambos são úteis como parâmetros indicadores para o início da terapia e na avaliação de sua eficácia. Além disto, a sua associação a testes de genotipagem para a identificação de mutações de resistência viral, pode auxiliar na indicação da conduta clínica mais adequada. No presente estudo, analisamos os valores da carga viral e taxas de linfócitos T CD4+ e CD8+ na avaliação do status imunológico de 25 crianças com indicação para a terapia antiretroviral, condicionando o regime terapêutico aos resultados do teste de genotipagem. A identificação dos subtipos virais foi feita por análise filogenética e a genotipagem incluiu a análise dos genes protease e transcriptase reversa do HIV-1. Dezoito amostras foram agrupadas no subtipo viral B e três no subtipo F1; cepas recobinantes também foram observadas, sendo uma BF, duas BD e uma DF. Dezoito crianças apresentaram mutações conferindo resistência viral aos inibidores da transcriptase reversa análogos de nucleosídeo e sete crianças apresentaram resistência aos inibidores não-análogos, com seis relatando resistência a nevirapina, delavirdina e efavirenz. Além disto, duas crianças, nas quais a terapia havia sido descontinuada dois a três anos antes da avaliação do teste de genotipagem, apresentaram as mutações K101E, K103N e G190A, conferindo resistência às três drogas. As mutações mais frequentes para o gene da transcriptase reversa foram observadas nos codons M41L, M184V e T215FY...

With antiretroviral therapy becoming more widely available nowadays. Human Immunodeficiency Virus type 1 resistance identification and monitoring of disease remains of great importance in adults and infected children. The major HIV-1 infection markers usually used for monitoring viral infection and disease course are CD4+ T cell counts or percentages and HIV viral load. Both of them are helpful indicating when to start therapy and evaluating its efficacy. Also, their association with genotyping tests to identify viral resistant mutations may help clinicians for the most adequate clinical conduct. In the present study, we assessed HIV-1 viral load and CD4+ and CD8+ T lymphocyte rates for the immunological status evaluation of 25 antiretroviral-treated children managing therapeutic regimens according to genotyping test results. Drug resistance evaluation was done using genotyping covering protease and reverse transcriptase genes. Additionally, all of the 25 vertically HIV-1 infected children were assessed for viral subtyping throughout phylogenetic analysis. Eighteen samples clustered at B subtype and three clustered at F1 subtype; one BF, two BD and one DF recobinant strains were also observed. Eighteen children presented, at least, one mutation conferring resistance to the nucleoside reverse transcriptase inhibitors, and seven children presented resistance to the non-nucleoside inhibitors, with six resistant to all three drugs, nevirapine, delavirdine, and efavirenz. In addition, two children in whom the therapy had been discontinued two to three years before testing presented K101E, K103N, and G190A mutations conferring resistance to the all three drugs. Reverse transcriptase gene mutations were more frequently observed at codons M41L, M184V, and T215FY. However, ten children presented and important number of viral resistance mutations, ranging from five to ten mutations, coferring resistance to at least four to up to eleven antiretroviral drugs...