Usefulness of rpoB Gene Sequencing for Identification of Afipia and Bosea Species, Including a Strategy for Choosing Discriminative Partial Sequences

Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability “hot spots” in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.     

A rare localization in right-sided endocarditis diagnosed by echocardiography: A case report

Background

Right-sided endocarditis occurs predominantly in intravenous drug users, patients with pacemakers or central venous lines and with congenital heart diseases. The vast majority of cases involve the tricuspid valve.

Case presentation

A case of a 31-year-old woman with intravenous drug abuse who had a right-sided vegetation attached to the muscular bundle of the right ventricle is presented. Transthoracic echocardiography revealed a vegetation in the right ventricular outflow tract. Transesophageal echocardiography clearly showed that the 1.8 cm vegetation was not adherent to the pulmonary valve but attached to a muscular bundle.

Conclusions

Our case points to an unusual location of right-sided endocarditis in intravenous drug users. It confirms that TTE remains an easy and highly sensitive first-line examination for the diagnosis of right-sided endocarditis.     

Evaluation of the lambda model for human postural control during ankle strategy

An accurate modeling of human stance might be helpful in assessing postural deficit. The objective of this article is to validate a mathematical postural control model for quiet standing posture. The postural dynamics is modeled in the sagittal plane as an inverted pendulum with torque applied at the ankle joint. The torque control system is represented by the physiological lambda model. Two neurophysiological command variables of the central nervous system, designated and , establish the dynamic threshold muscle at which motoneuron recruitment begins. Kinematic data and electromyographic signals were collected on four young males in order to measure small voluntary sway and quiet standing posture. Validation of the mathematical model was achieved through comparison of the experimental and simulated results. The mathematical model allows computation of the unmeasurable neurophysiological commands and that control the equilibrium position and stability. Furthermore, with the model it is possible to conclude that low-amplitude body sway during quiet stance is commanded by the central nervous system.     

Atypical Human Infections by Animal Trypanosomes

The two classical forms of human trypanosomoses are sleeping sickness due to Trypanosoma brucei gambiense or T. brucei rhodesiense, and Chagas disease due to T. cruzi. However, a number of atypical human infections caused by other T. species (or sub-species) have been reported, namely due to T. brucei brucei, T. vivax, T. congolense, T. evansi, T. lewisi, and T. lewisi-like. These cases are reviewed here. Some infections were transient in nature, while others required treatments that were successful in most cases, although two cases were fatal. A recent case of infection due to T. evansi was related to a lack of apolipoprotein L-I, but T. lewisi infections were not related to immunosuppression or specific human genetic profiles. Out of 19 patients, eight were confirmed between 1974 and 2010, thanks to improved molecular techniques. However, the number of cases of atypical human trypanosomoses might be underestimated. Thus, improvement, evaluation of new diagnostic tests, and field investigations are required for detection and confirmation of these atypical cases.

Key Learning Points

• The classical human trypanosomoses are human African trypanosomosis (HAT) or sleeping sickness, and Chagas disease, the Latin American human trypanosomosis.
• Atypical human infections caused by Trypanosoma species that normally are restricted to animals have been reported. These cases of atypical human trypanosomoses (a-HT) are mostly transient, but some require treatment and can be fatal.
• Only a few cases of a-HT have been fully confirmed, especially in Asia, leading to the hypothesis that the actual prevalence is probably underestimated.
• The detection of a case of a-HT should be based on observation of the parasite by direct microscopy. Evaluating/improving the diagnoses through serological and PCR assays would help in detecting and identifying atypical trypanosomosis infections in humans. These laboratory research and field activities are needed to evaluate the actual occurrence of atypical cases.

Top Five Papers

1. Verma A, Manchanda S, Kumar N, Sharma A, Goel M, et al. (2011) Trypanosoma lewisi or Trypanosoma lewisi-like infection in a 37-day-old infant. Am J Trop Med Hyg 85: 221–224.
2. Deborggraeve S, Koffi M, Jamonneau V, Bonsu FA, Queyson R, et al. (2008) Molecular analysis of archived blood slides reveals an atypical human Trypanosoma infection. Diagn Microbiol Infect Dis 61: 428–433.
3. Vanhollebeke B, Truc P, Poelvoorde P, Pays A, Joshi PP, et al. (2006) Human Trypanosoma evansi infection linked to a lack of apolipoprotein L-I. N Engl J Med 355: 2752–2756.
4. Joshi PP, Shegokar V, Powar S, Herder S, Katti R, et al. (2005) Human trypanosomiasis caused by Trypanosoma evansi in India: the first case report. Am J Trop Med Hyg 73: 491–495.
5. Howie S, Guy M, Fleming L, Bailey W, Noyes H, et al. (2006) A Gambian infant with fever and an unexpected blood film. PLoS Med 3: e355. doi:10.1371/journal.pmed.0030355.

     

Biosecurity measures in 48 isolation facilities managing highly infectious diseases

Biosecurity measures are traditionally applied to laboratories, but they may also be usefully applied in highly specialized clinical settings, such as the isolation facilities for the management of patients with highly infectious diseases (eg, viral hemorrhagic fevers, SARS, smallpox, potentially severe pandemic flu, and MDR- and XDR-tuberculosis). In 2009 the European Network for Highly Infectious Diseases conducted a survey in 48 isolation facilities in 16 European countries to determine biosecurity measures for access control to the facility. Security personnel are present in 39 facilities (81%). In 35 facilities (73%), entrance to the isolation area is restricted; control methods include electronic keys, a PIN system, closed-circuit TV, and guards at the doors. In 25 facilities (52%), identification and registration of all staff entering and exiting the isolation area are required. Access control is used in most surveyed centers, but specific lacks exist in some facilities. Further data are needed to assess other biosecurity aspects, such as the security measures during the transportation of potentially contaminated materials and measures to address the risk of an "insider attack."     

Rapid and sensitive high-performance liquid chromatographic method for simultaneous determination of retinol, α-tocopherol, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human plasma with photodiode-array ultraviolet detection

A new rapid and sensitive high-performance liquid chromatographic method using 0.5 ml of plasma has been developed for the simultaneous determination of retinol (vitamin A), α-tocopherol (vitamin E), 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃. The eluate was monitored with a photodiode-array detector with two fixed wavelengths (267 nm for vitamin D, 292 nm for α-tocopherol and retinol). For all compounds, including internal standards, the method provides extraction recoveries greater than 81%. Detection limits were equal to or lower than 1.5 μg/l for the 4 vitamins. Linearity of standards was excellent (r>0.999 in all cases). Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was 7.7 for all compounds and the inter-day-assay C.V. was <9.2% except for the lower concentrations of 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₂ and α-tocopherol (10.8, 11.8 and 11.9, respectively). The important properties of the present method are its ease of use, its rapidity, since sample preparation was achieved in 15 min and all the compounds were eluted in less than 15 min, and its small sample volume required (=0.5 ml), which enables it to be used in pediatric practice.     

TNF-mediated toxicity after massive induction of specific CD8+ T cells following immunization of mice with a tumor-specific peptide

We immunized mice with antigenic peptide P815E, which is presented by H-2K(d) and recognized by tumor-specific CTL raised against P815 tumor cells. This peptide is encoded by the ubiquitously expressed gene MsrA and carries a mutated residue conferring tumor specificity. Unexpectedly, we observed a severe toxicity occurring in the early hours after the third injection, resulting in the death of most mice within 24 h. The toxic syndrome was reminiscent of TNF-induced shock, and the sera of ill mice contained high levels of TNF. Toxicity was prevented by injection of neutralizing anti-TNF Abs, confirming the involvement of TNF. Depletion of CD8+ T cells could also prevent toxicity, and ex vivo experiments confirmed that CD8+ lymphocytes were the major cellular source of TNF in immunized mice. Tetramer analysis of the lymphocytes of immunized mice indicated a massive expansion of P815E-specific T cells, up to >60% of circulating CD8+ lymphocytes. A similar toxicity was observed after massive expansion of specific CD8+ T cells following immunization with another P815 peptide, which is encoded by gene P1A and was injected in a form covalently linked to an immunostimulatory peptide derived from IL-1. We conclude that the toxicity is caused by specific CD8+ lymphocytes, which are extensively amplified by peptide immunization in a QS21-based adjuvant and produce toxic levels of TNF upon further stimulation with the peptide. Our results suggest that immunotherapy trials involving new peptides should be pursued with caution and should include a careful monitoring of the T cell response.     

Effects of HIV-1 Nef on retrograde transport from the plasma membrane to the endoplasmic reticulum

HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb₃, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, βCOP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed .     

Identification and cellular localization of the actin-binding protein ABP-120 from Entamoeba histolytica

Several actin-binding proteins participate in the morphological changes that occur during amoeboid movement. The gene encoding one of these proteins, the gelation factor ABP-120, was identified and characterized from trophozoites of Entamoeba histolytica . The sequence contains 2574 nucleotides, with an open reading frame of 858 amino acids, giving a protein of 93 kDa belonging to the spectrin family. The N-terminal domain of ABP-120 from E. histolytica revealed a consensus site for actin binding homologous to the actin-binding sites of ABP-120 of Dictyostelium discoideum , α-actinin and spectrin. Analysis of the central domain revealed the presence of four repeats of a 73-amino-acid motif constituting 31% of the protein. In addition, a stretch of 105 amino acids was highly divergent when compared with the C-terminal domain of D. discoideum ABP-120. This sequence showed short motifs that are homologous to microtubule-binding domains. We found that ABP-120 from E. histolytica binds to F-actin. In addition, upon motility of the parasite, this protein localized in the pseudopod and the uroid region, implying a role for ABP-120 in movement and capping of surface receptors in E. histolytica .     

Gelatinase B is involved in the in vitro wound repair of human respiratory epithelium

Following epithelial injury, extracellular matrix undergoes imposing remodelings. We examined the contribution of matrix metalloproteinases, gelatinases A and B, in an in vitro wound repair model of human respiratory epithelium. Confluent human surface respiratory epithelial (HSRE) cells cultured from dissociated surface cells of human nasal polyps were chemically injured. Over the next 3 to 5 days, cells migrated onto the injured area to repair the circular wound. Repair kinetics of these wounds was monitored until wound closure occurred. Gelatinolytic activities were analysed in culture supernates and in cell protein extracts derived from repairing migratory and non repairing stationary cells. Small amounts of gelatinase A were expressed by HSRE cells, and variations of this gelatinase remained very weak for the time of the wound repair. In contrast, gelatinase B was upregulated during the wound repair process, with a maximum peak observed at wound closure. A marked gelatinase B activation occurred only in cells involved in the repair process. Gelatinase B was localized in some migratory basal cells, recognized by an anti-cytokeratin 14 antibody and located around the wound. We could not detect any gelatinase A in repairing or in stationary HSRE cells. Addition of the 6-6B monoclonal antibody, known to inhibit gelatinase B activation, to the culture medium during the repair process resulted in a dose-dependent decrease of the wound repair speed. These results suggest that gelatinase B, produced by epithelial cells, actively contributes to the wound repair process of the respiratory epithelium. © 1996 Wiley-Liss, Inc.