Identification of Histoplasma capsulatum from culture extracts by real-time PCR

We designed and tested a real-time LightCycler PCR assay for Histoplasma capsulatum that correctly identified the 34 H. capsulatum isolates in a battery of 107 fungal isolates tested and also detected H. capsulatum in clinical specimens from three patients that were culture positive for this organism.     

Stimulation of T-helper cell gamma interferon and immunoglobulin G responses specific for Babesia bovis rhoptry-associated protein 1 (RAP-1) or a RAP-1 protein lacking the carboxy-terminal repeat region is insufficient to provide protective immunity against virulent B. bovis challenge

Rhoptry-associated protein 1 (RAP-1) is a targeted vaccine antigen for Babesia bovis and Babesia bigemina infections of cattle. The 60-kDa B. bovis RAP-1 is recognized by antibodies and T lymphocytes from cattle that recovered from infection and were immune to subsequent challenge. Immunization with native or recombinant protein was reported to reduce parasitemias in challenged animals. We recently reported that the NT domain of B. bovis RAP-1 contained immunodominant T-cell epitopes, whereas the repeat-rich CT domain was less immunostimulatory for T lymphocytes from cattle immune to B. bovis. The present study was therefore designed to test the hypothesis that the NT region of RAP-1, used as a vaccine with interleukin-12 and RIBI (catalog no. R-730; RIBI Immunochem Research, Inc., Hamilton, Mont. [now Corixa, Seattle, Wash.]) adjuvant to induce a type 1 response, would prime calves for antibody and T-helper cell responses comparable to or greater than those induced by full-length RAP-1 containing the C-terminal repeats. Furthermore, a type 1 immune response to RAP-1 was hypothesized to induce protection against challenge. Following four inoculations of either recombinant full-length RAP-1 or RAP-1 NT protein, RAP-1-specific immunoglobulin G (IgG) titers, T-lymphocyte proliferation, and gamma interferon production were similar. Similar numbers of NT region peptides were recognized. However, in spite of the presence of strong RAP-1-specific IgG and CD4(+)-T-lymphocyte responses that were recalled upon challenge, neither antigen stimulated a protective immune response. We conclude that successful priming of calves with recombinant RAP-1 and adjuvants that elicit strong Th1 cell and IgG responses is insufficient to protect calves against virulent B. bovis challenge.     

In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.     

Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection

The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.     

Passive intranasal monoclonal antibody prophylaxis against murine Pneumocystis carinii pneumonia

Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model of Pneumocystis carinii pneumonia, we were able to demonstrate protection against an airborne challenge with P. carinii by intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and human P. carinii organisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection with P. carinii.     

Intranasal vaccination induces protective immunity against intranasal infection with virulent Francisella tularensis biovar A

The inhalation of Francisella tularensis biovar A causes pneumonic tularemia associated with high morbidity and mortality rates in humans. Exposure to F. tularensis usually occurs by accident, but there is increasing awareness that F. tularensis may be deliberately released in an act of bioterrorism or war. The development of a vaccine against pneumonic tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. Vaccine models for F. tularensis in inbred mice would facilitate investigations of the protective mechanisms and significantly enhance vaccine development. Intranasal vaccination with the attenuated live vaccine strain (LVS) of F. tularensis reproducibly protected BALB/c mice, but not C57BL/6 mice, against intranasal and subcutaneous challenges with a virulent clinical isolate of F. tularensis biovar A (NMFTA1). The resistance of LVS-vaccinated BALB/c mice to intranasal NMFTA1 challenge was increased 100-fold by boosting with live NMFTA1 but not with LVS. The protective response was specific for F. tularensis and required both CD4 and CD8 T cells. The vaccinated mice appeared outwardly healthy for more than 2 months after NMFTA1 challenge, even though NMFTA1 was recovered from more than half of the vaccinated mice. These results show that intranasal vaccination induces immunity that protects BALB/c mice from intranasal infection by F. tularensis biovar A.     

Detection and Identification of Actinobacillus pleuropneumoniae Serotype 5 by Multiplex PCR

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10² CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.     

An observational study of variation in GPs' out-of-hours emergency referrals.

Out-of-hours organisations are responsible for the care of patients 70% of the time, and their GPs act as gatekeepers to secondary care services. This observational study identifies the variations in GPs' out-of-hours referral rates to secondary care and factors that could explain these variations. One hundred and forty-nine GPs who worked in one UK general practice out-of-hours cooperative which served 19 practices with 167 000 registered patients. Data on patients who accessed the out-of-hours service over 3 years (2001-2004) were examined. Factors thought to be predictors of variation in referral rates were investigated using logistic regression analysis. There was a fivefold difference in referral rates between the lowest and highest referring quartiles of GPs (OR [odds ratio] = 4.56, CI [confidence interval] = 3.86 to 5.38). The sex (female) of the clinician, the time of the consultation (11 pm to 7 am), and the place of the consultation (home visit) accounted for some, but not all, of the increased referral rates. A doctor working out-of-hours disproportionately influences the fate of the patient, the number of hospital admissions, and extra costs to the health service. There is a need for follow-up studies to investigate the factors associated with referral behaviour, and how the variation relates to patient factors and the resources available. These findings could be used when planning the staffing of out-of-hours services to optimise appropriate care and minimise patients' exposure to unnecessary intrusive and expensive hospital care.     

Neurobehavioral Symptoms and Family Functioning in Traumatically Brain-Injured Adults

Traumatic brain injury (TBI) often results in a myriad of symptoms across physical, cognitive, and neurobehavioral domains. Despite inherent limitations associated with physical or cognitive impairments, the extant literature suggests that neurobehavioral symptoms tend to be the most distressing symptoms for the family and are more strongly related to poor outcome for the patient. The Neuropsychology Behavior and Affect Profile (NBAP) along with the General Functioning subscale of the Family Assessment Device (FAD-GF) and the Perceived Stress Scale were administered to 153 family members of persons who had sustained a TBI. The results provide new normative data and statistical support for the NBAP as a promising measure of neurobehavioral symptomatology following TBI. The correlation of.54 (p <.01) between FAD-GF and Full Scale NBAP scores provides powerful support for the hypothesis that family dysfunction is related to the presence of neurobehavioral symptoms in the patient. NBAP domains of Depression, Inappropriateness, Pragnosia, and Indifference appear most strongly related to family functioning and also bear a significant relationship to caregiver stress level and patient unemployment, whereas injury severity had little impact on either family functioning or neurobehavioral symptoms. The findings reinforce the significance of neurobehavioral symptoms and fortify their proposed link to family dysfunction post-TBI.     

Reproducibility of interpretation of the test for antibody-coated bacteria in urinary sediment.

The direct immunofluorescence technique for detecting antibody-coated bacteria in urinary sediment is felt to be useful in distinguishing infection of the kidney from infection of the bladder. An independent, blind multiple-reading system was used to measure interobserver variability in the evaluation of slides of urinary sediments for antibody-coated bacteria. Three independent observers agreed unanimously on first reading in 88% of 253 specimens. When compared with the majority opinion, the sensitivity and specificity of an individual reading were 91 and 95%, respectively.