RESUMO
Brazilian purpuric fever (BPF) is a highly fatal pediatric disease that may follow an episode of purulent conjunctivitis caused by a virulent clone of Haemophilus influenzae biogroup aegyptius (Hae). Oral rifampin prophylaxis, by eliminating carriage of the BPF clone in children with conjunctivitis, may prevent onset of the systemic disease. A test to detect the BPF clone directly from eye swabs could identify those in need of prophylaxis. This is a preliminary report of a rapid dot immunoassay performed on a "flow-through" cartridge that was developed for use under field conditions. The test is based upon recognition of a unique epitope of the 25-kDa pilin protein on the surface of BPF clone cells by a monoclonal antibody. With 36 laboratory-maintained cultures of Hae (15 clone isolates and 21 others), sensitivity of the assay was 67% and specificity was 95%. When fimbrial-enriched (25-kDa+) phenotypes of five false-negative clone strains were prepared for use as test antigens, sensitivity rose to 100%. Evaluation of the immunoassay under field conditions is necessary to prove its efficacy.
Assuntos
Infecções por Haemophilus/microbiologia , Haemophilus influenzae/isolamento & purificação , Immunoblotting/métodos , Conjuntivite Bacteriana/microbiologia , Humanos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Brazilian purpuric fever (BPF) is a recently described fatal pediatric disease caused by systemic infection with Haemophilus influenzae biogroup aegyptius. Previous studies have shown that all H. influenzae biogroup aegyptius strains isolated from BPF cases and case contacts share several unique phenotypic and genotypic characteristics that differentiate them from other H. influenzae biogroup aegyptius strains isolated from conjunctivitis cases in Brazil. One key characteristic of this BPF clone is reactivity in a BPF-specific monoclonal antibody enzyme-linked immunosorbent assay. We have purified and partially characterized a pilin, referred to as the 25-kilodalton (kDa) protein. Aggregates of this protein contain a heat-labile epitope which is recognized by a monoclonal antibody used in the BPF-specific enzyme-linked immunosorbent assay. The protein has a molecular weight of approximately 25,000, is insoluble in most detergents, and fractionates with outer membrane vesicles after LiCl extraction. Biochemical analysis of the 25-kDa protein shows it to have an amino acid composition similar but not identical to that of the H. influenzae type b pilin. The sequence of 20 N-terminal amino acids of the 25-kDa protein shows almost complete homology with the N terminus of the H. influenzae type b pilin and the types 1 and P pilins of Escherichia coli. Transmission electron microscopic analysis of the purified protein shows the presence of filamentous structures similar in morphology to those of H. influenzae pili. Reactivity between the 25-kDa protein and the BPF-specific monoclonal antibody is demonstrated by Western blotting (immunoblotting) and colloidal gold-enhanced immunoelectron microscopy. Hemadsorption analysis shows that expression of this protein is associated with increases in piliated cells and enhanced binding of these cells to human erythrocytes. These studies indicate that expression of the 25-kDa protein is a characteristic unique to the BPF clone and suggest that this protein plays a role in the pathogenesis of BPF.
Assuntos
Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Fímbrias Bacterianas , Haemophilus influenzae/química , Adsorção , Aminoácidos/análise , Anticorpos Monoclonais , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Fímbrias , Infecções por Haemophilus/etiologia , Haemophilus influenzae/patogenicidade , HumanosRESUMO
Cepas de H. aegyptus isoladas em surtos de Febre Purpúrica Brasileira (FPB) no Brasil, foram caracterizadas pelo método de aglutinaçäo em lâmina utilizando um anti-soro produzido com cepa de H. aegyptius isolada de cultura de sangue de paciente com FPB. Através desse método foi possível identificar cepas de H. aegyptius responsáveis por surtos de conjuntivite com características antigênicas iguais às cepas isoladas de FPB. A sensibilidade e especificidade da soroaglutinaçäo em lâmina foi de 97,7% e 89,6% respectivamente, podendo ser utilizado como método de triagem em estudos de conjuntivites purulentas, para detectar cepas invasivas de H. aegyptius associada a FPB, possibilitando assim a implantaçäo de medias que ampliem a eficiência na prevençäo e na vigilância epidemiológica da doença
Assuntos
Humanos , Pré-Escolar , Criança , Febre/microbiologia , Infecções por Haemophilus/epidemiologia , Doença Aguda , Fatores Etários , Brasil/epidemiologia , Conjuntivite Bacteriana/etiologia , Conjuntivite Bacteriana/microbiologia , Febre/epidemiologia , Haemophilus influenzae/classificação , Haemophilus influenzae/isolamento & purificação , Testes de Aglutinação/métodosRESUMO
Strains of H. aegyptius isolated during outbreak of Brazilian Purpuric Fever (BPF) in Brazil were characterized antigenically by slide agglutination test utilizing antiserum produced with a H. aegyptius strain isolated from blood culture from a patient with BPF. By means of this method, it were identified H. aegyptius strains responsible for outbreaks of conjunctivitis with identical antigenic characteristics to strains isolated from BPF. The sensitivity and specificity of slide seroagglutination test was 97.7% and 89.6% respectively; therefore this assay was efficient to be used as a screening method in the studies of purulent conjunctivitis for detecting high risk populations for BPF, and to implement measures that will increase the efficiency of epidemiologic surveillance.
Assuntos
Febre/microbiologia , Infecções por Haemophilus/epidemiologia , Doença Aguda , Fatores Etários , Testes de Aglutinação/métodos , Brasil/epidemiologia , Criança , Pré-Escolar , Conjuntivite Bacteriana/etiologia , Conjuntivite Bacteriana/microbiologia , Febre/epidemiologia , Haemophilus influenzae/classificação , HumanosAssuntos
Infecções por Haemophilus/microbiologia , Haemophilus influenzae/classificação , Púrpura/etiologia , Técnicas de Tipagem Bacteriana , Brasil , Centers for Disease Control and Prevention, U.S. , Infecções por Haemophilus/complicações , Haemophilus influenzae/isolamento & purificação , Humanos , Estados UnidosAssuntos
Conjuntivite Bacteriana/diagnóstico , Infecções por Haemophilus/diagnóstico , Púrpura/diagnóstico , Testes de Aglutinação , Brasil , Conjuntivite Bacteriana/etiologia , Infecções por Haemophilus/complicações , Haemophilus influenzae/genética , Haemophilus influenzae/isolamento & purificação , Humanos , Técnicas Imunoenzimáticas , Testes de Fixação do Látex , Programas de Rastreamento , Púrpura/etiologia , Sensibilidade e EspecificidadeRESUMO
Brazilian purpuric fever (BPF) is a recently recognized fulminant pediatric disease characterized by fever, with rapid progression to purpura, hypotensive shock, and death. BPF is usually preceded by purulent conjunctivitis that has resolved before the onset of fever. Both the conjunctivitis and BPF are caused by Haemophilus influenzae biogroup aegyptius (formerly called H. aegyptius). Isolates from 15 BPF cases, mainly from blood or hemorrhagic cerebrospinal fluid, case-associated isolates from 42 persons in towns where BPF cases occurred, and control strains from 32 persons in towns without BPF cases were characterized biochemically, genetically, and epidemiologically. Results indicated that a single clone was responsible for all BPF cases identified in six Brazilian towns from 1984 through 1986. All of 15 (100%) case strains were the same clone as was 1 of 32 (3%) control strains (P = less than 10(-8). Isolates of the clone were preferentially intrarelated by DNA hybridization (99% relatedness, hydroxyapatite method at 60 and 75 degrees C) and were separable from other H. influenzae biogroup aegyptius strains (approximately 90% relatedness at 60 degrees C and 82% relatedness at 75 degrees C). All isolates of the BPF clone and no other strains contained a 24-megadalton plasmid of restriction endonuclease type 3031, were of a single multilocus enzyme mobility type, were of a single sodium dodecyl sulfate-polyacrylamide gel electrophoresis type, and were in one of two ribosomal DNA restriction patterns. All BPF clone isolates reacted with monoclonal antibodies produced from a case strain; only 3 of 62 (5%) other strains reacted with this monoclonal antibody. Ninety percent of BPF clone strains and 27% of other strains were relatively resistant to sulfamethoxazole-trimethoprim.