Low Rates of Pseudomonas aeruginosa Misidentification in Isolates from Cystic Fibrosis Patients

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n = 21), Stenotrophomonas maltophilia (n = 15), and Inquilinus limosus (n = 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phenotypic features should be evaluated further by additional phenotypic or genotypic identification techniques.The accurate identification of Pseudomonas aeruginosa is a critical component of cystic fibrosis (CF) patient management. Once established within CF lungs, P. aeruginosa is rarely eradicated, leading to increased treatment requirements and an accelerated decline in pulmonary function, quality of life, and life expectancy (10, 13, 27). Emerging evidence indicates that aggressive antipseudomonal therapy at the time of initial acquisition may eliminate P. aeruginosa, preventing the development of chronic infection for months or even years (37). Similarly, separating patients with P. aeruginosa from other CF patients may reduce the spread of multiple-antibiotic-resistant strains capable of person-to-person transmission (16). Such strategies are contingent upon the early and correct identification of these organisms (30).While there is much emphasis on misidentifying P. aeruginosa as another species (39), less attention is paid to falsely identifying other species as P. aeruginosa. Nevertheless, accurate identification of P. aeruginosa is important, as this may avoid prolonged and sometimes unnecessary antibiotic treatments, which could select for other antibiotic-resistant pathogens (6). Similarly, in CF clinics where cohort isolation is practiced as an infection control measure, false identification could mean exposure of the CF patient to potentially transmissible bacteria (2, 17, 28, 33).While most clinical strains of P. aeruginosa are easily identified, respiratory isolates from patients with CF can present a taxonomic challenge (15, 24). Phenotypic identification of P. aeruginosa from patients with CF is often complicated by slow growth, auxotrophic metabolic activity, loss of pigment production, multiple antibiotic resistance, atypical colonial morphology, and development of mucoid exopolysaccharide (14, 25). Commercial identification platforms are also considered unreliable (18, 21, 39). Moreover, CF respiratory secretions may contain other nonfermenting gram-negative bacilli, such as Achromobacter, Stenotrophomonas, and Burkholderia species, which can further impede the identification of P. aeruginosa (29, 32, 35, 39).Although several molecular strategies have been developed recently (1, 35, 39), most clinical microbiology laboratories still identify P. aeruginosa by traditional phenotypic techniques. However, there are few published data describing the frequency at which bacterial species in CF sputum are falsely identified as P. aeruginosa by phenotypic methods. In this study, we used P. aeruginosa-specific duplex real-time (PAduplex) PCR assays, phenotypic analysis, amplified rRNA gene restriction analysis (ARDRA), and partial 16S rRNA gene sequence analysis to assess the rate and extent of misidentification of P. aeruginosa isolates in CF sputum by Australian clinical microbiology laboratories.     

Serum TABM produced during anterior chamber-associated immune deviation passively transfers suppression of delayed-type hypersensitivity to primed mice

Injection of soluble protein antigen into the anterior chamber of the eye of primed mice induces anterior chamber-associated immune deviation (ACAID) which is manifested by suppression of delayed-type hypersensitivity (DTH) to the antigen. Recently, we found that ACAID induced in primed mice also results in a rapid rise in serum of soluble T lymphocyte-derived proteins specific for nominal antigen (TABM). Here, we demonstrate that serum TABM induced in primed mice during ACAID will transfer the suppression of DTH to mice primed to the same antigen. Sera from TNP-BSA-primed mice that received an anterior chamber injection of TNP-BSA, but not BSA alone, suppressed the DTH response to TNP when injected into other TNP-BSA-primed mice. Sera absorbed with Sepharose beads conjugated with either anti-TCR C(alpha), anti-TCR C(beta), anti-TABM or TNP-BSA did not contain TNP-specific TABM and did not transfer suppression of DTH. These results suggest that the antigen-specific, TCR C(alphabeta)+ TABM that appear in serum during ACAID are able to confer on or amplify the capacity of sensitized T cells to suppress DTH. We believe this to be the first demonstration of an in vivo immunologic function that is specifically associated with TABM produced in vivo.      

Coelacanth genome sequence reveals the evolutionary history of vertebrate genes

The coelacanth is one of the nearest living relatives of tetrapods. However, a teleost species such as zebrafish or Fugu is typically used as the outgroup in current tetrapod comparative sequence analyses. Such studies are complicated by the fact that teleost genomes have undergone a whole-genome duplication event, as well as individual gene-duplication events. Here, we demonstrate the value of coelacanth genome sequence by complete sequencing and analysis of the protocadherin gene cluster of the Indonesian coelacanth, Latimeria menadoensis. We found that coelacanth has 49 protocadherin cluster genes organized in the same three ordered subclusters, alpha, beta, and gamma, as the 54 protocadherin cluster genes in human. In contrast, whole-genome and tandem duplications have generated two zebrafish protocadherin clusters comprised of at least 97 genes. Additionally, zebrafish protocadherins are far more prone to homogenizing gene conversion events than coelacanth protocadherins, suggesting that recombination- and duplication-driven plasticity may be a feature of teleost genomes. Our results indicate that coelacanth provides the ideal outgroup sequence against which tetrapod genomes can be measured. We therefore present L. menadoensis as a candidate for whole-genome sequencing.     

IL-10-driven immunoglobulin production by B lymphocytes from IgA-deficient individuals correlates to infection proneness

In search for a possible explanation of the phenotypic heterogeneity in IgA deficiency, we studied the function of B cells from IgA-deficient (IgAd) individuals. Two groups of IgAd individuals, one frequently infected and one clinically apparently healthy, as well as normal controls, were studied. Peripheral blood mononuclear cells (PBMC) and B cells from IgAd individuals and controls were cultured with Staphylococcus aureus Cowan I strain and with anti-CD40 MoAb presented on the CD32-transfected fibroblast cell line in the presence of IL-10. In this experimental system PBMC and B cells from the infection-prone IgAd individuals produced only minute amounts of IgA. In contrast, PBMC and B cells from healthy IgAd subjects secreted significantly more IgA1 and IgA2 in comparison with infection-prone IgAd patients (P < 0.05). These data suggest that the abnormalities of B cell differentiation in IgAd could be of heterogeneous origin. Thus, whereas in healthy IgAd subjects IgA production may be efficiently up-regulated in vitro by addition of IL-10 to CD40-activated B cell culture, the corresponding B cell differentiation does not occur in infection-prone IgAd patients. These observations provide a conceptual framework for phenotypic heterogeneity in IgAd subjects.     

A clinical study assessing the tolerability and biological effects of infliximab, a TNF-alpha inhibitor, in patients with advanced cancer

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) is an important regulator of the chronic inflammation contributing to tumour progression. Infliximab, an anti-TNF-alpha monoclonal antibody was investigated in this trial of patients with advanced cancer. The primary objectives were to determine the safety profile and biological response of infliximab in a cancer population. Clinical response was a secondary objective. PATIENTS AND METHODS: Forty-one patients received infliximab at 5 mg/kg (n = 21) or 10 mg/kg (n = 20) i.v. at 0 and 2 weeks and then every 4 weeks. Post-treatment samples were measured for changes in plasma and serum TNF-alpha, CCL2, IL-6 and C-reactive protein (CRP). RESULTS: Infliximab was well tolerated with no dose-limiting toxic effects. At both doses of infliximab, neutralisation of serum TNF-alpha was observed after 1 h while plasma CCL2, IL-6 and serum CRP were decreased 24 and 48 h following infliximab administration. Seven patients experienced disease stablisation (range 10-50+ weeks). There was no evidence of disease acceleration in any patient. CONCLUSIONS: Infliximab treatment was safe and well tolerated in patients with advanced cancer. There was evidence of biological activity with baseline TNF-alpha and CCL2 being correlated with infliximab response.     

Feeding during acute diarrhea as a risk factor for persistent diarrhea

Dietary intake during diarrhea in children less than three years of age was estimated from information recorded on illustrated dietary forms used by children's caretakers during the first week of illness in a prospective community-based study of diarrheal diseases in Lima, Peru. The frequency of consumption and the amount consumed of food groups and selected commonly consumed foods were analyzed by the final duration of the diarrheal episode. Cereals were less frequently consumed during the acute phase of diarrheal episodes that ultimately became persistent (>14 days'duration), apparently shortening the duration of the episode by one day (median duration of four days in children not consuming vs three days in children consuming cereals during diarrhea, p <0.02 Kaplan-Meier logrank test). Only roots and tubers (mainly potatoes) were consumed in greater quantity during episodes that became persistent. There was no evidence that consumption of breast milk or non-maternal milk was associated with an alteration in diarrheal duration. This study provides further evidence of the beneficial effects of continuing feeding during diarrhea using foods available at the home level, especially cereals, which are commonly used in the diet of young children.