Human Serum Activities against Hemophilus influenzae, Type b

Humoral immunity to Hemophilus influenzae, type b was studied in normal human adults by means of assays for serum bactericidal and opsonizing activities against the organism and for passive hemagglutinating activity using erythrocytes sensitized with polyribophosphate, the type-specific capsular antigen. Hemagglutinating activity was detectable in about 60% of the 114 sera tested. Serum bactericidal and opsonizing activities were found in all sera tested; the levels in some sera, however, were quite low. The antibacterial activities were due not only to antibodies directed against the polyribophosphate capsule but also to antibodies that appear to be directed against somatic antigens. Type b strains differed in their susceptibility to the antisomatic antibodies of particular sera but were uniformly sensitive to anticapsular antibody.     

Pathophysiology of hemolysis in infections with Hemophilus influenzae type b.

The capsular polysaccharide of Hemophilus influenzae type b, polyribosyl ribitol phosphate (PRP), is released from growing organisms during human infection and can be found in body fluids. It binds to untreated erythrocytes. Many patients with invasive infections with this organism develop significant hemolysis, but the mechanism has been unclear. We have found that PRP binds to human erythrocytes in vivo. PRP-coated erythrocytes have a shortened circulation time in mice, but do not lyse spontaneously or fix complement. PRP-coated erythrocytes exposed to antiserum to H. influenzae type b are undamaged in the absence of complement, but are rapidly and effectively lysed in the presence of an intact complement system both in vitro and in vivo in mice. PRP-coated red cells are taken up by liver and spleen. Antiserum to PRP increases hepatic uptake of PRP-coated red cells more than splenic, and appears to induce intravascular, complement-mediated hemolysis, as well as extravascular hemolysis. Patients with invasive infection develop hemolysis when circulating PRP and antibody to PRP are present simultaneously. PRP can sometimes be detected on patient erythrocytes when free PRP is present in serum, but this is an inconsistent finding. The hemolytic anemia that occurs during human infection with H. influenzae type b may be due to absorption of PRP to red cells and immune destruction of sensitized erythrocytes. The process requires an intact complement system; both complement-mediated cell lysis and extravascular hemolysis contribute to red cell destruction.     

Circulating polyribophosphate in Hemophilus influenzae, type b meningitis. Correlation with clinical course and antibody response.

In systemic infections caused by Hemophilus influenzae, type b, the capsular polysaccharide, polyribophosphate, is released into the circulation. Polyribophosphate was quantitated in serial serum and cerebrospinal fluid samples from 45 children with H. influenzae, type b meningitis by means of a radiolabeled antigen-binding inhibition assay. Polyribophosphate was regularly found in acute serum and cerebrospinal fluid samples and could be detected in unbound form for periods of 1-30 days after initiation of effective therapy. Complexes of polyribophosphate dissociable with acid and pepsin were detected in serum samples from 17 patients, in one case for a period of 145 days after hospitalization. Polyribophosphate levels and patterns of clearance were studied in relation to hospital course and antibody response. Patients with prolonged antigenemia had protracted fevers and severe neurological symptoms during hospitalization, frequently with focal complications.Antipolyribophosphate antibody responses were detected during the first 100 days of convalescence by radioimmunoassay in 79% of the patients studied, including 60% of the children 1 yr or less in age. The intensity of antibody response although clearly related to the age of the patient, was more reliably predicted by the efficiency of antigen clearance. Antibody responses were uniformly of low magnitude in patients with prolonged antigenemia, irrespective of age. Paients who failed to develop antibody to polyribophosphate after meningitis also exhibited impaired antigen clearance. These studies suggest that mechanisms necessary for clearance of polyribophosphate may influence the development and intensity of the humoral immune response and raise the possibility of developmental deficiencies in the clearance system in infants and children.     

Induction of streptomycin resistance in sensitive Hemophilus influenzae by extracts containing desoxyribonucleic acid from resistant Hemophilus influenzae

Resistance to streptomycin, of a degree exceeding 1000 µg. per ml., has been induced in sensitive strains of Hemophilus influenzae by exposure for 10 minutes to desoxyribonucleic acid-containing extracts isolated from a strain of type b Hemophilus influenzae which had emerged resistant to 1000 µg. of streptomycin per ml. DNA is essential for the process which brings out this change; the reaction can be prevented by destruction of the DNA with crystalline desoxyribonuclease.The resistant trait which is created in this way is heritable. The nature of the process which induces resistance is similar in all respects to the reaction which induces heritable changes in type specificity of H. influenzae. These results offer another example of the gene-like action of highly specific DNA's. The pattern of resistance brought out in a bacterial population exposed to the DNA-containing, resistance-inducing extract, is similar to that which occurs when emergence of resistance of H. influenzae to streptomycin follows the selection by streptomycin of spontaneously occurring resistant mutants. The change in a bacterial cell from average susceptibility to streptomycin to resistance to 1000 µg. of streptomycin per ml. can occur in a single step.     

Ampicillin-Induced Morphological Alterations of Haemophilus influenzae Type b

Inhibitory levels of ampicillin induced filamentation of growing Haemophilus influenzae ATCC 19418 within 30 min. Filaments became swollen and interrupted by regular periodic saccular outpouchings along the major axis. The degree of filamentation was dependent upon ampicillin concentration and time.     

Initial treatment of Hemophilus influenzae infections in children

Life-threatening pediatric infections in which H. influenzae is the potential pathogen include meningitis, septic arthritis, cellulitis and epiglottis. Until the pathogen is identified, chloramphenicol should be part of the initial hospital therapy. This agent is effective against ampicillin-susceptible and ampicillin-resistant H. influenzae strains, and it reliably penetrates the central nervous system.     

Capsular typing of Haemophilus influenzae with a DNA probe.

We describe the use of a DNA probe for genotyping clinical isolates of Haemophilus influenzae. The probe, containing capsulation genes, differentiates between the six Haemophilus serotypes in a Southern blotting procedure. It also hybridizes with a distinctive pattern to DNA from capsule-deficient mutants of serotype b strains, while failing to hybridize to DNA from typical clinical isolates of non-serotypable H. influenzae. The probe can thus resolve issues of serotyping uncertainty such as arise, for example, when capsulate strains are found to have lost reactivity with serotyping reagents after storage or transmission from one laboratory to another. The probe has proved useful in the evaluation of Haemophilus infections in infants following administration of H. influenzae type b vaccine. In an illustrative example, the probe was used to resolve serotyping ambiguity in a case of Haemophilus bacteraemia in a vaccine recipient, providing compelling evidence that the organism responsible was neither type b nor derived from a type b strain. The widespread introduction of vaccines against H. influenzae type b disease will increase the importance of the precise identification of strains infecting immunized children. This need can only be met by the development of 'gold standards' such as capsulation gene probes.     

Susceptibility of Haemophilus influenzae Type b to Cefatrizine, Ampicillin, and Chloramphenicol

The susceptibility of 269 isolates of Haemophilus influenzae type b to cefatrizine (BL-S640), ampicillin, and chloramphenicol was evaluated by disk diffusion susceptibility tests, using a modified Bauer-Kirby method. Broth dilution susceptibility tests were performed on 88 of these isolates, including all isolates resistant by disk to cefatrizine or ampicillin. Six of the isolates were resistant by disk to cefatrizine (zone size, <16 mm), four were resistant to ampicillin (zone size, <19 mm), and none were resistant to chloramphenicol (zone size, <17 mm). Only two of the six isolates of H. influenzae that were resistant to cefatrizine by disk were resistant to more than 4 mug of drug per ml. The four organisms resistant to ampicillin on disk were resistant, when tested by the broth method, to >128 mug/ml. These four ampicillin-resistant H. influenzae were susceptible to <4 mug of cefatrizine per ml. The two isolates resistant to >4 mug of cefatrizine per ml were susceptible to 0.5 and 2 mug of ampicillin, respectively, per ml. The activity of cefatrizine appears to be comparable in vitro to ampicillin against H. influenzae.     

Hemophilus influenzae f cellulitis with bacteremia, peritonitis, and pleuritis in an adult with nephrotic syndrome.

Hemophilus influenzae f was responsible for cellulitis with bacteremia, pleuritis, and peritonitis in an adult patient with the nephrotic syndrome. The patient rapidly responded to ampicillin. H influenzae f has previously been rarely found to cause pleuritis and bacteremia, but has not been reported as a cause of cellulitis or primary peritonitis. Patients with the nephrotic syndrome are prone to serious infection with encapsulated bacteria. The relative frequency of infection with the various encapsulated bacteria most likely parallels that of colonization by these organisms.     

Susceptibility of Haemophilus influenzae Type b to Rifampin and Sulfisoxazole

A total of 100 and 97% of Haemophilus influenzae type b strains from major infections were susceptible, respectively, to levels of rifampin and sulfisoxazole attainable in saliva. It is theoretically feasible to eliminate Haemophilus influenzae from the nasopharynx with these drugs.     

2014年成都某院流感嗜血杆菌的临床分布及耐药分析

目的：探讨该院临床分离的流感嗜血杆菌分布及抗菌药物的耐药情况。方法以该院2014年1～12月所有已筛查合格且培养为阳性的下呼吸道标本1895份、血液类标本666份、尿道分泌物标本102份,共2663份标本分离出的流感嗜血杆菌438株为研究对象,采用头孢硝基噻吩纸片测定其β‐内酰胺酶,用纸片扩散法（K‐B 法）测定其对8种抗菌药物的敏感性,利用WHONET5．6软件对试验数据进行统计分析。结果438株流感嗜血杆菌中98．6％来自住院患者,1．4％来自门诊;儿童分离株有176株,占40．18％;老年患者分离株194株,占44．29％;下呼吸道标本来源434株,占99．09％。对复方磺胺甲噁唑耐药率最高,达82．90％,氨苄西林和四环素的耐药率分别为43．40％、29．00％;而头孢呋辛、头孢曲松、环丙沙星、阿奇霉素、亚胺培南的耐药率均在2．00％以下。β‐内酰胺酶的阳性率为43．40％,在氨苄西林耐药菌株中有1．57％的菌株β‐内酰胺酶检测阴性。儿童分离株对氨苄西林的耐药率和产酶率（42．40％、40．43％）和成人分离菌株对氨苄西林的耐药率及产酶率（44．00％、44．00％）没有明显差异。结论流感嗜血杆菌主要分离自下呼吸道标本中;以儿童和老年患者为主,第二、三代头孢菌素类与阿奇霉素可作为临床首选药物,β‐内酰胺酶是该菌对氨苄西林耐药的主要机制。     

Immunization with Haemophilus influenzae type b polysaccharide vaccine at 18 and 24 months of age: evidence of decreased immunogenicity.

In order to assess the immunogenicity of a two-dose regimen of Haemophilus influenzae type b polysaccharide vaccine, the immune response to vaccine given at both 18 and 24 months of age was compared to the response to a single dose at 24 months of age. Following immunization at 24 months of age, the geometric mean antibody concentration of children previously immunized at 18 months (0.53 micrograms/ml) was significantly lower than that of children who received a single dose of vaccine at 24 months (1.03 micrograms/ml; p = 0.03). A four-fold rise in antibody concentration was demonstrated in 31% of children who had received two immunizations and 53% of children immunized for the first time at 24 months of age (p = 0.02). These results suggest that the administration of a dose of plain polysaccharide vaccine at 18 months of age blunted the response to reimmunization at 24 months of age. A subgroup of children who failed to respond to one or two doses of plain polysaccharide vaccine were immunized with polysaccharide-diphtheria toxoid conjugate vaccine. The majority of these children developed a significant rise in antibody concentration in response to conjugate vaccine.     

Expression of crossreactive idiotypes by human antibodies specific for the capsular polysaccharide of Hemophilus influenzae B.

Human antibodies specific, for polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hemophilus influenzae b, were studied using idiotypic analysis. Antisera were prepared against purified F(ab')2 anti-PRP from two unrelated adults, H.H. and P.T. After repeated absorption with IgG myeloma proteins and with PRP-absorbed normal human Ig and donor Ig, anti-idiotypic (anti-Id) sera were obtained that specifically reacted with anti-PRP antibodies. Anti-IdHH and anti-IdPT reciprocally crossreacted with H.H. and P.T. anti-PRP antibodies and F(ab')2 fragments, and also reacted with the serum anti-PRP antibodies from three additional adults unrelated to P.T. and H.H. Both anti-Id sera partially inhibited anti-PRP paratopes but not anti-tetanus toxoid paratopes. PRP did not inhibit anti-Id recognition of shared or crossreactive idiotypic (CRI) determinants. Naturally occurring and PRP immunization-induced anti-PRP antibodies expressed CRI. While CRI titer increased after immunization, the increase was usually less than the rise in total anti-PRP antibody. Quantitative differences in CRI expression were also apparent between natural and immunization-induced H.H. and P.T. anti-PRP antibodies as shown by their differential inhibitability by anti-Id. Our data demonstrate that anti-PRP antibodies from five unrelated adults express CRI determinants that are probably distant from the PRP combining site. Naturally occurring and immunization-induced anti-PRP antibodies share CRI and therefore appear to be clonally related, although immunization apparently induces the expression CRI-negative antibodies as well. These results, taken with previous studies showing restricted and identical anti-PRP isoelectric focusing spectrotypes in unrelated adults, suggest that some PRP-specific V domains are structurally conserved and probably germ-line encoded.     

Treatment of otitis media caused by Hemophilus influenzae: evaluation of three antimicrobial regimens.

Ninety young children with otitis media, proven by tympanocentesis culture to be due to Hemophilus influenzae, were treated in a prospective double-blind study with one of three antimicrobial regimens: ampicillin, erythromycin ethylsuccinate, or erythromycin ethylsuccinate with concomitant trisulfapyrimidines. Results of efficacy evaluation indicated that the combination was as effective as ampicillin and statistically superior to erythromycine alone. Of the strains of H influenzae identified, 15/9 were typable with 12/9, type b. In vitro sensitivity tests indicated that the strains were sensitive to the amtimicrobials studied in clinically attainable levels; however, comparison of individual efficacy ratings with sensitivity results indicated that some children in each treatment group failed to respond as predicted.     

Phenotypic and Genetic Variation in the Susceptibility of Haemophilus influenzae Type b to Antibodies to Somatic Antigens

Haemophilus influenzae type b (H.i.b) has been investigated with respect to phenotypic and genetic variations resulting in differential susceptibility to bactericidal antibody. Previous studies had shown that after growth in infected rats or in dialysate of rat serum, H.i.b strain Eag became more resistant to the bactericidal activity of antisomatic antibody. We now report that a similar phenotypic shift occurs when strain Eag is incubated with dialysate of human serum, that the increased resistance is to antibodies against determinants in the lipopolysaccharide not for the somatic antigens generally, and that most strains of H.i.b undergo the shift.To assess genetic differences in exposed antigens, a panel of 13 H.i.b isolates from cerebrospinal fluid were analyzed with cross-adsorbed antisera. Seven different patterns were found that could be accounted for through the variable expression of six antigens. These ranged from infrequent (found on 1:13 strains) to common (10:13 strains). At least four were somatic rather than capsular determinants; the most common (antigen 1) was contained in lipopolysaccharide.The epidemiologic relevance of the genetic variations was explored using pairs of isolates from two children who had had two documented infections with H.i.b. In both cases the isolates varied in somatic antigen expression. The strains from one patient differed in the expression of antigen 1. The isolates from the other were indistinguishable in sub-typing for the six classified antigens, but differed in the expression of an additional antigen identified by use of the patient's serum.     

Active Immunization with Lipopolysaccharide Pseudomonas Antigen for Chronic Pseudomonas Bronchopneumonia in Guinea Pigs

Chronic respiratory infection with Pseudomonas aeruginosa is a leading clinical problem among patients with cystic fibrosis. Because antimicrobial agents are usually ineffective in eradicating these infections, additional therapeutic or prophylactic measures should be considered. In this study, an experimental guinea pig model of chronic Pseudomonas aeruginosa bronchopneumonia was utilized to determine whether active immunization with lipopolysaccharide (LPS) P. aeruginosa antigen may favorably influence the course of this infection. Experimental pneumonia was established by tracheobronchial instillation of suspensions of microscopic agar beads, which were impregnated with viable P. aeruginosa. After 4 wk of infection, the geometric mean (reciprocal) passive hemagglutinating Pseudomonas antibody titer was 185±1.3, and lungs contained 16.8±4 × 10³ colony-forming units Pseudomonas/ml of lung homogenate. Pseudomonas immunization, given prior to a 4-wk infection, resulted in significantly higher passive hemagglutinating titers (474±1.4; P < 0.05), lower numbers of viable Pseudomonas in lung tissues (2.4±0.6 × 10³; P < 0.01), and reduced histopathology in lungs. In contrast, providing Pseudomonas immunization to animals 2 wk after pulmonary infection was established, offered no apparent benefit. Likewise, no protection was afforded by prophylactic immunization with a non-Pseudomonas LPS antigen (Escherichia coli J5 vaccine). Using a Raji cell assay, modified to detect circulating immune complexes in vaccinated and infected guinea pig sera, there was no evidence that active immunization increased the frequency of circulating immune complexes in infected guinea pigs.It is concluded that prophylactic immunization with Pseudomonas LPS antigen may confer protection from subsequent Pseudomonas bronchopneumonia, but that immunization during established infection is not beneficial.     

Rapidly increasing prevalence of beta-lactamase-nonproducing, ampicillin-resistant Haemophilus influenzae type b in patients with meningitis

A total of 395 Haemophilus influenzae strains from 226 Japanese institutions participating in the Nationwide Surveillance Study Group for Bacterial Meningitis were received from 1999 to 2002. All strains were analyzed by PCR to identify the resistance genes, and their susceptibilities to beta-lactam agents were determined. Of these strains, 29.1% were beta-lactamase nonproducing and ampicillin (AMP) susceptible (BLNAS) and lacked all resistance genes; 15.4% were beta-lactamase producing and AMP resistant and had the bla(TEM-1) gene; 30.6% were beta-lactamase nonproducing and AMP resistant (low-BLNAR) and had a Lys-526 or His-517 amino acid substitution in ftsI encoding PBP 3; 13.9% were beta-lactamase nonproducing and AMP resistant (BLNAR) and had an additional substitution of Thr-385 in ftsI; 9.1% were amoxicillin-clavulanic acid resistant (BLPACR I) and had the bla(TEM-1) gene and a Lys-526 or His-517 amino acid substitution in ftsI; and 1.8% showed resistance similar to that of the BLPACR I group (BLPACR II) but had bla(TEM-1) gene and ftsI substitutions, as was the case for the BLNAR strains. All but three strains were serotype b. The prevalence of BLNAR strains has increased rapidly: 0% in 1999, 5.8% in 2000, 14.1% in 2001, and 21.3% in 2002. The MICs at which 90% of BLNAR isolates were inhibited were as follows: AMP, 16 micro g/ml; cefotaxime, 1 micro g/ml; ceftriaxone, 0.25 micro g/ml; and meropenem, 0.5 micro g/ml. All of these values were higher than those for the BLNAS counterpart strains. The relatively wide distributions of the beta-lactam MICs for BLNAR strains presumably reflect variations in ftsI gene mutations. Pulsed-field gel electrophoresis suggested the rapid spread of specific H. influenzae type b strains throughout Japan. Expedited vaccination, rapid identification, and judicious antibiotic use could slow their spread.