Tuberculosis-associated hemophagocytic lymphohistiocytosis with subsequent unmasking cryptococcal immune reconstitution inflammatory syndrome (IRIS) in an HIV-negative man.

A 22-year-old HIV-negative man from Ghana was diagnosed with severe hemophagocytic lymphohistiocytosis (HLH) induced by multiorgan tuberculosis with peritoneal, hepatic, pericardial, myocardial, pleural, pulmonary, and bone manifestation. His body mass index was 12.9 m2/kg. Bioptic material of a peritoneal biopsy grew M. tuberculosis, sensitive to all first-line antituberculous drugs. HLH resolved with antituberculous therapy, without additional anti-inflammatory therapy being given. The initial CT scan of his brain was normal. After 5 months of antituberculous treatment, he developed a paralysis of the left arm. A cerebral MRT showed ring-enhanced lesions. Blood cultures and lumbar puncture revealed Cryptococcus neoformans var. grubi. The HIV test was repeatedly negative. Antituberculous treatment was continued for a total of 9 months, and additional treatment with antifungal therapy was established. He recovered fully after 14 months of antifungal treatment.

Laboratory Experience with the Liaison Analyzer in the Diagnosis of Clostridium Difficile-Associated Diarrhea.

BACKGROUND: Chemiluminescent or enzyme-linked fluorescent immunoassays are commonly used to diagnose Clostridium difficile-associated diarrhea. METHODS: The LIAISON analyzer (DiaSorin, Italy) was compared to miniVIDAS (bioMérieux, France) and, furthermore, to culture of toxigenic strains. In total, 249 native stool samples were analyzed. Sensitivities, specificities, and positive and negative predictive values were investigated. Furthermore, performance under routine conditions was assessed. RESULTS: The glutamate dehydrogenase chemiluminescent immunoassay (GDH-CLIA) assay revealed a high sensitivity and negative predictive value. The toxins A&B assays exhibited approximately the same low sensitivity and high specificity. Technical drawbacks experienced with the LIAISON analyzer in 48% of the analyses considerably delayed the time to the first diagnostic report and interfered with laboratory routine workflow. CONCLUSION: The analytical performance of the investigated platforms should be reflected in the context of implementation into the laboratory workflow.

Blood sampling as a cause of anemia in a general ICU - a pilot study.

OBJECTIVE: The objective of our pilot study was to evaluate the influence of daily phlebotomy on patients' haemoglobin levels from our general intensive care unit. METHODS: We prospectively enrolled 35 patients who did not present with acute haemorrhage or developed it during the study period. For each patient we recorded: the diagnosis, age, sex, haemoglobin, hematocrit, SOFA and APACHE II score, blood volume drawn in standardized vials, number of blood tests ordered per day, fluid balance per day, number of ICU days. The collected data were analyzed using the linear regression model, paired t-test, receiver operating characteristic curves, and descriptive analysis. Statistical analysis was performed with SPSS v.17 trial version (IBM, NY, USA). RESULTS: The mean volume of blood drawn per day was 18.1 (SD ± 14.4) ml and the number of blood tests was 3.8 (SD ± 1.75) per day. On univariate linear regression analysis both the blood volume drawn daily (p = 0.04) and the number of blood tests per day (p = 0.009) correlated with a drop in mean haemoglobin concentration. The difference in the mean value of haemoglobin at admission and discharge correlated with overall mortality (p = 0.03). The sensitivity of admission haemoglobin equal to 10.6 g/dL in predicting mortality was 82.4% with a specificity of 50%, (p = 0.019, AUC = 0.732). CONCLUSIONS: We evidenced the predictive power of blood sampling and number of blood tests done on haemoglobin concentration. Besides the main objective of the study we noticed that the difference in the mean value of haemoglobin at admission and discharge correlated with overall mortality. Considering that blood sampling contributes to anemia among ICU patients, we should limit the daily tests undertaken, to the tests absolutely necessary for guiding our therapy.

Mapping of possible laminin binding sites of Y. pestis plasminogen activator (Pla) via phage display.

We tried to determine amino acid motifs of Y. pestis plasminogen activator (Pla) involved in laminin binding. We selected heptamer peptides using a random phage library which was tested against immobilised laminin. Two sequences seemed to inhibit Pla mediated laminin binding of E. coli and exhibited a strong laminin binding capacity revealing a competition with Pla for the same laminin binding site. The motifs are also involved in plasminogen activation because they caused fibrinolysis on fibrin films. The patterns were localised outside the putative surface-exposed loops (Kukkonen et al., 2001).

Mobility of the Yersinia High-Pathogenicity Island (HPI): transfer mechanisms of pathogenicity islands (PAIS) revisited (a review).

Horizontal gene transfer (HGT) plays a key role in the evolution of bacterial pathogens. The exchange of genetic material supplies prokaryotes with several fitness traits enhancing their adaptive response to environmental changes. Pathogenicity islands (PAIs) represent an important and in most cases already immobilized subset of the different vehicles for HGT. Encoding several virulence factors PAls represent a major contribution to bacterial pathogenicity. Nonetheless, the transfer mechanisms of PAIs still remain elusive. We summarise the currently available data regarding the major ways of genetic mobilisation with a focus on the transfer of the Yersinia High-Pathogenicity Island (HPI).

Identification of laminin-binding motifs of Yersinia pestis plasminogen activator by phage display.

Yersinia pestis plasminogen activator (Pla), a surface virulence factor contributes to the highly invasive nature of the pathogen by binding various tissue matrix components. In this study we characterised the laminin-binding site(s) of Pla via phage display and alanine-scanning mutagenesis. Previously we isolated 18 different heptamer peptide sequences from a phage display library with biopanning on laminin, and have shown that two of them with sequences of WSLLTPA or YPYIPTL completely inhibited laminin binding of a Pla-expressing recombinant Escherichia coli strain. These phages themselves strongly bound laminin in an ELISA assay utilising horseradish peroxidase-labelled anti-M 13 antibody. In the present study, with the application of synthetic peptides, a 55% and a 33% inhibition was demonstrated using WSLLTPA and YPYIPTL, respectively. Peptide pattern alignment showed two homologous regions for WSLLTPA and one for YPYIPTL inside the Pla sequence. Amino acids were changed for alanine in one of the WSLLTPA regions and in the YPYIPTL region in order to prove the role of the LTP/PTL motifs in laminin binding. Of the four constructed mutants, the triple mutant L65A-T66A-L67A in the WSLLTPA region and the double mutant G178A-L179A in the YPYIPTL region decreased the laminin binding capacity of the Pla-expressing recombinant E. coli by about 50%.

Intracellular signalling and cytoskeletal rearrangement involved in Yersinia pestis plasminogen activator (Pla) mediated HeLa cell invasion.

Yersinia pestis, the etiologic agent of plague is a highly invasive organism being able to invade non-phagocytic epithelial cells. Its plasminogen activator (Pla), encoded by the pPCP1 plasmid plays a pivotal role in internalisation of bacteria by HeLa cells. The aim of this study was to analyse the intracellular signalling processes and cytoskeletal rearrangement events associated with invasion. Wortmannin caused a 50% decrease of invasiveness at 50nM concentration pointing to the involvement of phosphatidyl-inosinol-4 kinase (PtINs4). Pre-treatment with staurosporin, a potent inhibitor of protein kinases (PKs) and with genistein, a specific tyrosine kinase inhibitor decreased the number of internalised bacteria about seven-fold and two-fold, respectively, indicating the involvement of PKs including tyrosine kinases in Pla-mediated internalisation. Cytochalasin D, an actin polymerisation inhibitor, C3 exoenzyme of Clostridium botulinum, a specific inhibitor of small GTPase Rho, and NDGA, a 5-lipoxygenase inhibitor also involved in Rho activation strongly reduced the number of internalised bacteria revealing the role of cytoskeletal events in the invasion process. All the tested inhibitors changed the invasion but not the adhesion pattern of the Pla producing recombinant strain. Actin rearrangement could also be visualised also with rhodamin-phalloidin staining.

Species-specific peptide ligands for the detection of Bacillus anthracis spores.

Currently available detectors for spores of Bacillus anthracis, the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B. anthracis spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B. anthracis spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B. anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B. anthracis. These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B. anthracis spores. We envision that these peptides can be used as sensors in economical and portable B. anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.

Peptide ligands that bind selectively to spores of Bacillus subtilis and closely related species.

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of Bacillus subtilis. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of B. subtilis and its most closely related species, Bacillus amyloliquefaciens, and slightly weaker binding to spores of the closely related species Bacillus globigii. These three species comprise one branch on the Bacillus phylogenetic tree. We did not detect peptide binding to spores of several Bacillus species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of B. subtilis. The sequence Asn-His-Phe-Leu-Pro was used to identify B. subtilis proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.