Evaluation of an Internet-based decision-support system for applying the ATS/CDC guidelines for tuberculosis preventive therapy.

Preventive therapy for patients infected with tuberculosis (TB) remains an important component of TB control. To guide physicians in applying preventive therapy, the American Thoracic Society and Centers for Disease Control (ATS/CDC) developed guidelines based on PPD reactivity and on pretest probability of infection. The guidelines have become complex, and many clinicians find them challenging to apply. The authors developed a computerized decision-support system to assist clinicians in applying the ATS/CDC guidelines. This tool, published on the World Wide Web using hypertext markup language, delivers patient-specific recommendations based on physician-delivered patient-specific information. Four local TB experts derived eight TB infection scenarios and validated the web-based tool, which was tested for effectiveness using general internal medicine residents, randomly divided into two groups. Group A (n = 12) used the web-based tool and group B (n = 17) used pre-existing understanding of the guidelines and/or written resources to determine the need for preventive therapy in the case scenarios. Group A correctly used therapy in 92/96 possible cases (95.8%), group B in only 77/136 (56.6%) (p < 0.001). Group A required a mean of three mouse-clicks and 1.5 minutes per scenario to reach their choices, and they rated the web-based tool both intuitive and effective. These data demonstrate that a computer-based decision-support system for applying TB treatment guidelines can be delivered over the Internet and provide an efficient and effective resource for clinicians.

Therapy for ventilator-associated pneumonia.

Pneumonia is a serious complication of mechanical ventilation. Pneumonia occurs despite the best efforts at prevention. Multiple methods available to prevent ventilator-associated pneumonia are reviewed, and ventilation-associated pneumonia (VAP) is divided into early versus late onset. The authors discuss the organisms associated with each of these situations, the empiric antibiotic choices, and specific issues related to antibiotic therapy such as resistance, pharmcodynamics, tissue penetration, and types of modifications necessary in empiric choice when the cause of VAP is identified.

Infection of primary human bronchial epithelial cells by Haemophilus influenzae: macropinocytosis as a mechanism of airway epithelial cell entry.

Nontypeable Haemophilus influenzae is an exclusive human pathogen which infects the respiratory epithelium. We have initiated studies to explore the interaction of the nontypeable H. influenzae strain 2019 with primary human airway epithelial cells by electron and confocal microscopy. Primary human airway cell cultures were established as monolayers on glass collagen-coated coverslips or on semipermeable membranes at an air-fluid interface. Scanning electron microscopy indicated that bacteria adhered to nonciliated cells in the population. The surface of infected cells showed evidence of cytoskeletal rearrangements manifested by microvilli and lamellipodia extending toward and engaging bacteria. Confocal microscopic analysis demonstrated that infection induced actin polymerization with an increase in cortical actin as well as evidence of actin strands around the bacteria. Transmission electron microscopic analysis showed lamellipodia and microvilli surrounding organisms, as well as organisms adherent to the cell surface. These studies also demonstrated the presence of bacteria within vacuoles inside of airway cells. Confocal microscopic studies with Texas red-labeled dextran (molecular weight, 70,000) indicated that H. influenzae cells were entering cells by the process of macropinocytosis. These studies indicate that nontypeable H. influenzae can initiate cytoskeletal rearrangement within human airway epithelium, resulting in internalization of the bacteria within nonciliated human airway epithelial cells by the process of macropinocytosis.

Characterization of the type 3 fimbrial adhesins of Klebsiella strains.

The Klebsiella pneumoniae fimbrial adhesin, MrkD, mediates adherence to the basolateral surfaces of renal and pulmonary epithelia and to the basement membranes of tissues. Although all isolates possessing the MrkD adhesin mediate the agglutination, in vitro, of erythrocytes treated with tannic acid, the mrkD gene is not conserved within species. The ability of a plasmid-borne mrkD gene product to mediate binding to type V collagen is associated frequently with strains of K. oxytoca and rarely with strains of K. pneumoniae. In K. pneumoniae, the MrkD adhesin is located within a chromosomally borne gene cluster and mediates binding to collagen types IV and V. The plasmid-borne determinant, mrkD1P, and the chromosomally borne gene, mrkD1C, are not genetically related. Some strains of enterobacteria possess a mrkD1C allele that is associated with hemagglutinating activity but does not bind to either type IV or type V collagen.

Patients with an abnormal chest radiograph and latent tuberculosis.

Patients with latent tuberculosis characterized by a positive tuberculin (purified protein derivative) skin test and radiographic evidence of fibronodular changes or silicosis are at increased risk for the development of active tuberculosis. Before preventive therapy is initiated, the radiographic abnormalities must be differentiated from those representing active disease. According to recommendations from the American Thoracic Society and the Centers for Disease Control and Prevention, patients with latent tuberculosis who exhibit fibronodular changes or silicosis on chest radiographs should be given either isoniazid alone for one year or the combination of isoniazid and rifampin for four months, preferably with pyrazinamide for the first two months. Patients with similar radiographic findings and sputum or culture evidence of active tuberculosis require full multidrug therapy.

Pathogenesis of respiratory infections and host defenses.

At the cellular level, the respiratory tract has a variety of defense mechanisms to prevent bacterial infection. Recent data have demonstrated that the respiratory epithelium plays a very active role in host defense. In this review we start by examining the respiratory epithelia and its function in mucociliary clearance, and extend our review to include its role in the secretion and regulation of inflammatory cytokines and production of antimicrobial factors. Furthermore, we examine how recent advances in understanding cystic fibrosis have provided useful insights into the pathogenesis of lower respiratory tract infection. In addition, we examine how two common respiratory pathogens, Streptococcus pneumoniae and Pseudomonas aeruginosa, subvert the defense mechanisms at the cellular level. Finally, we attempt to identify new or potential therapeutic approaches that have arisen from some of the insights into the pathogenesis of lower respiratory tract infections.

Adherence properties of an mrkD-negative mutant of Klebsiella pneumoniae.

The role of the mrkD gene in attachment by a type 3 fimbriate Klebsiella pneumoniae strain was further characterized. A clinical isolate, K. pneumoniae IA565, was found to contain two copies of the gene encoding the fimbrial subunit, mrkA, and one copy of the gene encoding the adhesin subunit, mrkD. One copy of mrkA was located on the bacterial chromosome, and the other copy was associated with mrkD and located on a plasmid. The plasmid-borne mrk gene cluster was lost when K. pneumoniae IA565 was subcultured serially in broth at 44 degrees C. The resulting mrkD-negative strain, designated K. pneumoniae IApc35, did not exhibit the following adherence characteristics associated with K. pneumoniae possessing MrkD-positive fimbriae: agglutination of tannic acid-treated human erythrocytes and attachment to trypsinized human buccal cells. However, K. pneumoniae IApc35 produced type 3 fimbriae that were composed of the characteristic 21.5-kDa major fimbrial subunit, were reactive with specific serum, and were visualized specifically by immunoelectron microscopy. K. pneumoniae IApc35 retained a copy of the mrkA gene on its chromosome. This mrkA-containing gene cluster could be complemented by a recombinant plasmid carrying only the mrkD gene, resulting in restoration of the K. pneumoniae IA565-like adhesive phenotype and demonstration of type 3 filament-associated MrkD subunits by using colloidal gold labeling and immunoelectron microscopy. These data indicate that K. pneumoniae may contain multiple copies of the mrk genes which may be present simultaneously on both plasmid and chromosomal DNAs and which may encode fimbriae with different binding specificities.

The type 3 fimbrial adhesin gene (mrkD) of Klebsiella species is not conserved among all fimbriate strains.

The type 3 fimbriae of enteric bacteria mediate agglutination, in vitro, of erythrocytes treated with tannic acid. The gene encoding the polypeptide, MrkD, that mediates this agglutination reaction was placed downstream of an inducible promoter, and the ability of MrkD alone to facilitate hemagglutination was determined. Although Escherichia coli transformants could be shown to produce the MrkD protein, hemagglutination did not occur in the absence of other mrk gene products. In addition, the MrkD polypeptide did not cross the bacterial outer membrane unless a fimbrial chaperone protein was also present. Analysis of the frequency of the mrkD gene within the genus Klebsiella indicated that this gene is conserved in strains of Klebsiella oxytoca but not in other fimbriate Klebsiella species. In the small number of strains of Klebsiella pneumoniae that do possess a related mrkD gene, this determinant could be found on a plasmid in one strain. The ability of type 3 fimbriate bacteria to adhere to type V collagen was found to be a function of a specific MrkD polypeptide. This adhesin is frequently found in strains of K. oxytoca but is rarely associated with the type 3 fimbriae of K. pneumoniae.

Emergence and persistence of Pseudomonas aeruginosa in the cystic fibrosis airway.

Colonization in the respiratory tracts of cystic fibrosis (CF) patients by mucoid Pseudomonas aeruginosa correlates with the progression of bronchial airway pathology. There is a direct correlation between the incidence of Pseudomonas colonization and age, clinical score, extent of pulmonary disease, severity of radiographic changes, and level of serum immunoglobulins. The central propensity to Pseudomonas colonization in patients with CF is not freely understood, but we discuss the acquisition and persistence of P aeruginosa in the CF airway. Elucidation of pathogenetic mechanisms of CF inflammatory airways disease is the first essential step to initiating novel therapies. It has been difficult to prove that the ability of P aeruginosa to adhere to the respiratory epithelium and provide selective advantage for this gram-negative bacillus over other potential pathogens for infection in the CF airway. However, flexible filaments (pili) extending from the Pseudomonas cell wall are thought to medicate epithelial cell adherence for nonmucoid P aeruginosa, and similarly, the gelatinous exopolysaccharide alginate produced by mucoid variants of P aeruginosa seems to be the adhesive to tracheal cells. Following the signal event of adherence, this bacterial pathogen competes successfully for iron cofactor and multiplies, releasing proteases with broad substrate specificities that dramatically alter the airway antiprotease screen, and the pathogen creates defects in local antibacterial defenses. Lung inflammation in CF is characterized by massive neutrophil infiltration. Although critical to host defense, neutrophils also cause progressive airway damage by release of bioactive lipids, oxygen metabolites, and granule enzymes such as hydrolases, myeloperoxidase (MPO), lysozyme, and neutral serine proteases. The necessarily circumscribed discussion that follows will focus narrowly on the host cell-derived factors (macrophages and neutrophils) proposed as important components in this pathogenetic scheme.

Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products.

We examined the role of Klebsiella fimbrial types 1 and 3 in mediating adherence to human buccal and tracheal cells and to lung tissue sections. We found that clinical isolates of Klebsiella pneumoniae producing type 3 fimbriae and Escherichia coli HB101 containing a recombinant plasmid encoding expression of Klebsiella type 3 fimbriae (pFK10) demonstrated increased adherence to tracheal cells, trypsinized buccal cells, and lung tissue sections, in contrast to nonfimbriate and to type 1 fimbriate bacteria. Adherence by type 3 fimbriate bacteria was inhibited by purified type 3 fimbriae and Fab fragments derived from type 3 fimbrial-specific polyclonal immunoglobulin G. Type 3 fimbriae mediated attachment to the basolateral surface of tracheal cells and to the basal epithelial cells and the basement membrane regions of bronchial epithelia. Using an E. coli transformant (pDC17/pFK52), which expresses nonadherent P fimbrial filaments, along with the type 3 fimbrial adhesin (MrkD), we demonstrated that type 3 fimbrial attachment to respiratory cells was attributable to the MrkD adhesin subunit. Subsequent experiments demonstrated that the epithelial target of the type 3 fimbrial adhesin was most likely a peptide molecule rather than a carbohydrate. The results of this study demonstrate that, in vitro, the Klebsiella type 3 fimbrial adhesin mediates adherence to human respiratory tissue.

Fimbrial types among respiratory isolates belonging to the family Enterobacteriaceae.

Bacterial attachment is believed to be an early step in gram-negative nosocomial pneumonia. The frequency of fimbria-associated adhesins among respiratory pathogens has not been studied in detail. In this study isolates belonging to the family Enterobacteriaceae, prospectively obtained from intensive care unit patients who were suspected of having nosocomial pneumonia, were examined for fimbria-associated adhesins. Type 3, P, type 1, and other fimbrial phenotypes were identified by specific hemagglutination and electron microscopy. The Klebsiella type 3 fimbrial phenotype was further characterized by using a monoclonal antibody. Also, both type 3 and Escherichia coli P fimbrial genotypes were detected by using DNA colony blot assays. The frequencies of genera or species isolated were as follows: Enterobacter (38.6%), Klebsiella (26.8%), Serratia (17.7%), E. coli (13%), and Proteus (5.2%). Isolates of Klebsiella oxytoca, K. pneumoniae, and Enterobacter cloacae most commonly possessed the type 3 fimbrial phenotype and genotype. The phenotype and genotype for E. coli P fimbriae (46.2 and 50%, respectively), a known pathogenic determinant in the urinary tract, were detected more frequently than expected. In addition, a previously unspecified hemagglutinin that was specific for porcine erythrocytes was almost uniformly expressed among isolates of Enterobacter aerogenes. Finally, the expression of the type 1 fimbrial phenotype was widely detected among the isolates tested but notably absent among K. oxytoca and Proteus mirabilis isolates. The frequency of the various fimbrial types identified suggests a role for these bacterial organelles in adherence to respiratory epithelia.

The immunoglobulin G subclass composition of immune complexes in cystic fibrosis. Implications for the pathogenesis of the Pseudomonas lung lesion.

It has been shown that pulmonary macrophage (PM) phagocytosis of Pseudomonas aeruginosa (PA) is inhibited in the presence of serum from cystic fibrosis (CF) patients colonized by Pseudomonas, and that these sera contain high concentrations of IgG2 antibodies. The goal of these studies was to investigate the role that IgG2-containing immune complexes (IC) play in this inhibition of both PM and neutrophil phagocytosis. We found that serum IgG2 concentrations were elevated significantly in CF patients with chronic PA colonization and that in selected sera from CF patients with chronic PA colonization (CF + IC, n = 10), the mean IC level was significantly elevated (2.90 +/- 0.22 mg/dl [SEM]). IgG2 comprised 74.5% of IgG precipitated in IC from CF + IC sera. An invitro phagocytic assay of [14C]PA uptake using CF + IC whole-sera opsonins confirmed that endocytosis by normal PM and neutrophils was significantly depressed. Removal of IC from CF + IC sera resulted in significantly decreased serum IgG2 concentrations without a significant change in the other subclass concentrations, and enhanced [14C]PA uptake by PM (26.6% uptake increased to 47.3%) and neutrophils (16.9% increased to 52.6%). Return of the soluble IgG2 IC to the original CF sera supernatants and the positive control sera resulted in return of the inhibitory capacity of the CF + IC sera. We conclude that immune sera from patients with chronic Pseudomonas infections characterized by elevated IgG2 subclass level functions poorly as an opsonin. In these individuals, IgG2 contributes significantly to circulating IC and removal of IC, matched by a simultaneous fall in IgG2, improves bacterial uptake by neutrophil and mononuclear phagocytes. IgG2 antibodies exert antiphagocytic effects by both direct inhibition and the formation of IC.

Pulmonary host defense: defects that lead to chronic inflammation of the airway.

Current knowledge of pulmonary host defense can help us to understand the unique relationship between CF patients and P. aeruginosa colonization of the lung. Subtle defects in CF host defense, such as those identified in mucociliary clearance and in the CF IgG opsonin, allow P. aeruginosa to persist. Not all the defects are attributable to the host. In several examples, the defects are induced by P. aeruginosa, presumably in an effort to maintain its foothold. The examples of the latter discussed here have included the effect of pseudomonas-derived products on mucociliary action, alpha 1 PI function, and the formation of ineffective IgG opsonins and immune complexes. Overall, P. aeruginosa is the cause of significant morbidity and, eventually, mortality in these patients. As we approach the identification of the genetic defect central to this disease, it is hoped that we will gain more insight into the pathogenesis of the P. aeruginosa lung lesion in CF and develop more effective ways of preventing P. aeruginosa colonization of the CF patients' lungs.

Nontuberculous mycobacterial lung disease. Substantiation of a less aggressive approach.

A nonsurgical, less aggressive, less toxic chemotherapeutic protocol for the management of nontuberculous mycobacterial (NTB) pulmonary infections has been uniformly applied to patients in our institution between 1972 and 1985. Forty-three nonimmunocompromised patients with active lung disease caused by Mycobacterium avium-intracellulare (MAI) (n = 26), M kansasii (n = 16), and M xenopi (n = 1) were identified retrospectively. Eighteen MAI patients were treated with three or four antituberculosis agents resulting in sputum conversion and clinical improvement in 12 (67 percent). Additionally, 11 out of 16 (69 percent) patients completing therapy or still undergoing therapy for persistent MAI disease, achieved sputum conversion and clinical improvement after prolonged therapy (3.6 +/- 0.5 years [SEM]). When M kansasii was identified as the etiologic agent, all patients were treated with four or fewer antituberculosis agents and 14 out of 16 patients (88 percent) achieved sputum conversion and clinical improvement throughout the follow-up period. We conclude that the use of three or four chemotherapeutic agents in the treatment of NTM lung disease provides an excellent probability of successful outcome even in MAI infections.