Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid-core peptide technology

A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid-core peptide technology was employed. Lipid-core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1-->LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side-chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side-chains increased immunogenicity. An increase in immunogenicity (enzyme-linked immunosorbent assay (ELISA) titres) of up to 100-fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freund's adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.

Microbiologia dos abscessos peritonsilares

OBJETIVO: O objetivo deste estudo é avaliar a microbiologia dos abscessos periamigdalianos. MATERIAIS E MÉTODOS: Trinta pacientes com diagnóstico de abscesso periamigdaliano, idade média de 24,2 anos, foram submetidos à punção na região periamigdaliana de maior abaulamento com aspiração do material purulento (volume maior que 3mL). O material foi separado para realização das culturas aeróbicas e anaeróbicas. RESULTADOS: Houve um índice de positividade das culturas de 86,7%. Em 23,3% aspirados houve crescimento apenas de bactérias aeróbicas ou facultativas, 3,3% apenas de bactérias anaeróbicas e por fim em 60% aspirados houve crescimento de bactérias aeróbicas e anaeróbicas. Um total de 69 bactérias foi isolado (34 aeróbios e 35 anaeróbios). Os aeróbios predominantes foram Streptococcus sp, sendo o Streptococcus pyogenes em 23% dos casos. Os anaeróbios predominantes foram Prevotella sp e Peptostreptococcus sp. Pacientes receberam antibiótico prévio em 63% dos casos. Neste grupo foram isolados 1,8 bactérias por aspirado, menor número que nos paciente que não utilizaram antibiótico (3,0 bactérias por aspirado). Não houve diferença significativa no tipo de bactéria isolada nestes dois grupos. CONCLUSÃO: Os abscessos periamigdalianos apresentam na maioria dos casos infecções polimicrobianas, sendo os organismos anaeróbicos agentes importantes. O número de agentes isolados é maior nos pacientes que não utilizaram antibioticoterapia prévia, mas o uso de antimicrobiano não interferiu no tipo de bactéria isolada.

Achado de bactérias selecionadas em crianças de Trinidad com doença amigdaliana crônica

Faringoamigdalite na população pediátrica é largamente tratada com antibióticos. OBJETIVO: Estudar a microflora presente na superfície e no núcleo de amígdalas após adenoamigdalectomia eletiva em crianças. MÉTODO: Amígdalas de 102 crianças de Trinidad foram prospectivamente estudadas por meio de culturas e identificações bacteriológicas feitas a partir de amostras das superfícies e núcleos de suas amígdalas entre 2005-2006. RESULTADOS: A partir de 360 amígdalas, foram isolados Streptococcus spp. (51,3%), Staphylococcus spp. (42,3%) e Gram-Negativos (6,4%). A identificação de estafilococos e estreptococos tanto na superfície quanto no núcleo foi semelhante (p>0,05). Encontramos mais (p<0,001) Streptococcus spp. nas superfícies (82,2%) do que nos núcleos (63,3%); a prevalência de estreptococos alfa-hemolíticos foi maior (p<0,001) do que aquela de estreptococos beta-hemolíticos nas superfícies (74,4% vs. 18,6%) do que nos núcleos (58,9% vs. 13,7%). Não houve concordância entre superfícies e núcleos com relação a estreptococos (p<0,0004) e estreptococos alfa-hemolíticos (p<0,007). Estreptococos beta-hemolíticos foram mais identificados (p<0,05) em crianças dentre 6-16 anos do que naquelas entre 1-5 anos de idade (31% e 23,8% vs 12,5% e 8%). A prevalência de S. pyogenes na superfície e no núcleo foi de (84,6% vs 70%) e (50,0% vs 25,0%) em crianças de maior faixa etária e crianças mais novas, respectivamente. Klebsiella spp. (6,6%, 2,2%), Proteus (4,4%, 4,4%) e Pseudomonas (4,4 %, 1,1%) cresceram nas superfícies e núcleos, respectivamente. CONCLUSÃO: As superfícies amigdalianas tinham mais estreptococos e estreptococos hemolíticos do que seus núcleos. Crianças mais velhas tiveram mais estreptococos beta-hemolíticos, e são altamente colonizadoras de S. pyogenes. Sugerimos estudos que investiguem os mecanismos de aderência estreptocócica em crianças de Trinidad.

Faringotonsilites em crianças: visão de uma amostra de pediatras e otorrinolaringologistas

As faringotonsilites agudas são infecções das vias aéreas superiores comuns na infância. OBJETIVO: Analisar opiniões e condutas de pediatras e otorrinolaringologistas do Estado de São Paulo em relação ao diagnóstico, tratamento e prevenção das faringotonsilites e suas complicações em crianças. MATERIAL E MÉTODOS: Selecionamos aleatoriamente 1370 pediatras e 1000 otorrinolaringologistas do Estado de São Paulo. Aos especialistas foi enviado questionário por correio. DESENHO DO ESTUDO: Estudo transversal. RESULTADOS: 95,8% dos pediatras e 91,5% dos otorrinos não solicitam rotineiramente exames para diagnóstico laboratorial das faringotonsilites agudas na criança. Os antimicrobianos mais prescritos pelos pediatras nas faringotonsilites bacterianas foram: penicilina por via oral durante 10 dias (33,6%) e penicilina benzatina em dose única (19,7%). Os antimicrobianos mais prescritos pelos otorrinos para tratamento foram: penicilina por via oral durante 10 dias (35,4%) e penicilina por via oral durante 7 dias (25,7%). A medida de prevenção das faringotonsilites bacterianas considerada muito eficaz por mais da metade dos pediatras e otorrinos foi a cirurgia de tonsilectomia. A faringotonsilite de repetição foi o principal motivo para os otorrinos indicarem cirurgia de tonsilectomia aos escolares e adolescentes (49,3% e 53,4%, respectivamente). CONCLUSÕES: É necessário uniformizar condutas de pediatras e otorrinos para diagnóstico e tratamento das faringotonsilites em crianças.

Synthesis, Antimicrobial and Antiproliferative Activity of Novel Silver(I) Tris(pyrazolyl)methanesulfonateand 1,3,5-Triaza-7-phosphadamantane Complexes

Five new silver(I) complexes of formulas [Ag(Tpms)] (1), [Ag(Tpms)-(PPh3)] (2), [Ag(Tpms)(PCy3)] (3), [Ag(PTA)][BF4] (4), and [Ag(Tpms)(PTA)] (5) {Tpms = tris(pyrazol-1-yl)methanesulfonate, PPh3 = triphenylphosphane, PCy3 = tricyclohexylphosphane, PTA = 1,3,5-triaza-7-phosphaadamantane) have been synthesized and fully characterized by elemental analyses, H-1, C-13, and P-31 NMR, electrospray ionization mass spectrometry (ESI-MS), and IR spectroscopic techniques. The single crystal X-ray diffraction study of 3 shows the Tpms ligand acting in the N-3-facially coordinating mode, while in 2 and 5 a N2O-coordination is found, with the SO3 group bonded to silver and a pendant free pyrazolyl ring. Features of the tilting in the coordinated pyrazolyl rings in these cases suggest that this inequivalence is related with the cone angles of the phosphanes. A detailed study of antimycobacterial and antiproliferative properties of all compounds has been carried out. They were screened for their in vitro antimicrobial activities against the standard strains Enterococcus faecalis (ATCC 29922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (SF37), Streptococcus sanguinis (SK36), Streptococcus mutans (UA1S9), Escherichia coli (ATCC 25922), and the fungus Candida albicans (ATCC 24443). Complexes 1-5 have been found to display effective antimicrobial activity against the series of bacteria and fungi, and some of them are potential candidates for antiseptic or disinfectant drugs. Interaction of Ag complexes with deoxyribonucleic acid (DNA) has been studied by fluorescence spectroscopic techniques, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA EB system on addition of Ag complexes shows that the fluorescence quenching of DNA EB complex occurs and compound 3 is particularly active. Complexes 1-5 exhibit pronounced antiproliferative activity against human malignant melanoma (A375) with an activity often higher than that of AgNO3, which has been used as a control, following the same order of activity inhibition on DNA, i.e., 3 > 2 > 1 > 5 > AgNO3 >> 4.

Evolution of genotypes of Group A streptococci from colonization and infection

Dissertação para obtenção do Grau de Doutor em Biologia

Empiema Torácico. Revisão Baseada em Três Casos Clínicos

O derrame parapneumónico caracteriza-se pela necessidade de um processo invasivo para a sua resolução e o empiema pela presença de pus na cavidade pleural. Em ambos os casos, o diagnóstico por TAC e o tratamento precoces resultando em menores morbilidade e mortalidade. São indicação para um tratamento invasivo os derrames loculados, os que ocupam mais de 50% do tórax, os que revelam coloração por Gram e exame cultural positivos, ou derrames com pH inferior a 7,20, glucose inferior a 60 mg/dl, e nível de DHL superior a três vezes o limite normal no soro. Estas características resultam da evolução através de três estádios dos derrames incorrectamente tratados: 1) exsudativo; 2) fibrino-purulento; 3) fibrótico. Dependendo do estádio evolutivo, a abordagem terapêutica varia entra toracentese terapêutica, colocação de drenagem torácica com ou sem instilação de fibrinolíticos, cirurgia toracoscópica vídeo-assistida e decorticação pulmonar. Os autores fazem uma revisão do estudo destas situações baseados em três casos clínicos com apresentações muito díspares: uma doente com empiema por Streptococcus pyogenes que faleceu rapidamente por hemoptise maciça; um doente com empiema resultante de pneumonia aguda ocorrida durante um voo de avião; uma doente com empiema e bacteriemia por Streptococcus pneumoniae conduzindo a diagnóstico até então desconhecido de infecção por VIH.

Epidemiological characterization, antimicrobial resistance and virulence mechanisms of human and animal streptococci

Dissertação para obtenção do Grau de Doutor em Biologia

Fasceite Necrosante e Otite Média Aguda

A fasceíte necrosante (FN) é uma doença rara em pediatria. Na maior parte dos casos surge como complicação de varicela, sendo a pele habitualmente a porta de entrada e o Streptococcus pyogenes o agente mais frequente. Os autores descrevem um caso clínico de uma criança de 8 anos, previamente saudável que, no contexto de uma otite média aguda medicada com um anti-inflamatório não esteróide (AINE), desenvolve uma infecção invasiva a Streptococcus 13 hemoliticus do grupo A.

Streptococcal acute pharyngitis

Acute pharyngitis/tonsillitis, which is characterized by inflammation of the posterior pharynx and tonsils, is a common disease. Several viruses and bacteria can cause acute pharyngitis; however, Streptococcus pyogenes (also known as Lancefield group A β-hemolytic streptococci) is the only agent that requires an etiologic diagnosis and specific treatment. S. pyogenes is of major clinical importance because it can trigger post-infection systemic complications, acute rheumatic fever, and post-streptococcal glomerulonephritis. Symptom onset in streptococcal infection is usually abrupt and includes intense sore throat, fever, chills, malaise, headache, tender enlarged anterior cervical lymph nodes, and pharyngeal or tonsillar exudate. Cough, coryza, conjunctivitis, and diarrhea are uncommon, and their presence suggests a viral cause. A diagnosis of pharyngitis is supported by the patient's history and by the physical examination. Throat culture is the gold standard for diagnosing streptococcus pharyngitis. However, it has been underused in public health services because of its low availability and because of the 1- to 2-day delay in obtaining results. Rapid antigen detection tests have been used to detect S. pyogenes directly from throat swabs within minutes. Clinical scoring systems have been developed to predict the risk of S. pyogenes infection. The most commonly used scoring system is the modified Centor score. Acute S. pyogenes pharyngitis is often a self-limiting disease. Penicillins are the first-choice treatment. For patients with penicillin allergy, cephalosporins can be an acceptable alternative, although primary hypersensitivity to cephalosporins can occur. Another drug option is the macrolides. Future perspectives to prevent streptococcal pharyngitis and post-infection systemic complications include the development of an anti-Streptococcus pyogenes vaccine.

In vitro cell activating properties of the composite ribosomal vaccine D53

D53 (RibomuntyR) is a composite vaccine made of immunogenic ribosomes from 4 bacterial species (Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pyogenes and Streptococcus pneumoniae) associated with a membrane proteoglycan from a non encapsulated strain of Klebsiella pneumoniae. D53 is a potent inducer of interleukin-1 production by mouse BALB/c spleen cells as shown by the C3H/HeJ thymocyte co-stimulation assay. Furthermore D53 triggers DNA synthesis by mouse spleen cells and induces the maturation of B lymphocytes into immunoglobulin secreting cells. Polyclonal B cell activation by D53 was readily achieved in the C3H/HeJ strain which is deficient in its response to E. coli lipopolysaccharide. The proliferative response to D53 was abrogated by removal of B cells from the spleen cell suspension, but it was not altered after depletion of T cells or adherent cells. D53 induced polyclonal B cell activation of spleen cells from athymic nude mice and from CBA/N mice. Each component of D53 induced polyclona B cell activation except ribosomes from Streptococcus pneumoniae. Each triggered Interleukin-1 synthesis except ribosomes from Klebsiella penumoniae. These in vitro properties may account for some of the in vivo immunostimulating properties of this composite vaccine.

Routine use of point-of-care tests: usefulness and application in clinical microbiology.

Point-of-care (POC) tests offer potentially substantial benefits for the management of infectious diseases, mainly by shortening the time to result and by making the test available at the bedside or at remote care centres. Commercial POC tests are already widely available for the diagnosis of bacterial and viral infections and for parasitic diseases, including malaria. Infectious diseases specialists and clinical microbiologists should be aware of the indications and limitations of each rapid test, so that they can use them appropriately and correctly interpret their results. The clinical applications and performance of the most relevant and commonly used POC tests are reviewed. Some of these tests exhibit insufficient sensitivity, and should therefore be coupled to confirmatory tests when the results are negative (e.g. Streptococcus pyogenes rapid antigen detection test), whereas the results of others need to be confirmed when positive (e.g. malaria). New molecular-based tests exhibit better sensitivity and specificity than former immunochromatographic assays (e.g. Streptococcus agalactiae detection). In the coming years, further evolution of POC tests may lead to new diagnostic approaches, such as panel testing, targeting not just a single pathogen, but all possible agents suspected in a specific clinical setting. To reach this goal, the development of serology-based and/or molecular-based microarrays/multiplexed tests will be needed. The availability of modern technology and new microfluidic devices will provide clinical microbiologists with the opportunity to be back at the bedside, proposing a large variety of POC tests that will allow quicker diagnosis and improved patient care.

Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer: reluctant response to antimonial treatment

We investigated the bacterial flora present in skin lesions of patients with chiclero's ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chiclero's ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chiclero's ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chiclero's ulcer.

Structure-function characterization and optimization of a plant-derived antibacterial peptide.

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.

Diagnostic des maladies infectieuses: place des "point of care tests" (POCT) [Use of POCT (point of care tests) in the diagnosis of infectious diseases]

Les POCT (point of care tests) ont un grand potentiel d'utilisation en médecine infectieuse ambulatoire grâce à leur rapidité d'exécution, leur impact sur l'administration d'antibiotiques et sur le diagnostic de certaines maladies transmissibles. Certains tests sont utilisés depuis plusieurs années (détection de Streptococcus pyogenes lors d'angine, anticorps anti-VIH, antigène urinaire de S. pneumoniae, antigène de Plasmodium falciparum). De nouvelles indications concernent les infections respiratoires, les diarrhées infantiles (rotavirus, E. coli entérohémorragique) et les infections sexuellement transmissibles. Des POCT, basés sur la détection d'acides nucléiques, viennent d'être introduits (streptocoque du groupe B chez la femme enceinte avant l'accouchement et la détection du portage de staphylocoque doré résistant à la méticilline). POCT have a great potential in ambulatory infectious diseases diagnosis, due to their impact on antibiotic administration and on communicable diseases prevention. Some are in use for long (S. pyogenes antigen, HIV antibodies) or short time (S. pneumoniae antigen, P. falciparum). The additional major indications will be community-acquired lower respiratory tract infections, infectious diarrhoea in children (rotavirus, enterotoxigenic E. coli), and hopefully sexually transmitted infections. Easy to use, these tests based on antigen-antibody reaction allow a rapid diagnosis in less than one hour; the new generation of POCT relying on nucleic acid detection are just introduced in practice (detection of GBS in pregnant women, carriage of MRSA), and will be extended to many pathogens