Implementation of MALDI-TOF MS technology for the identification of clinical isolates of Mycobacterium spp. in mycobacterial diagnosis.

A total of 243 clinical isolates of the Mycobacterium genus were studied, 143 and 100 using two protocols (Protocol v2 and Protocol v3, respectively) provided by the manufacturer. The overall correlation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the standard identification methods was 63.8 %. The rate of misidentification was 3.2 %, mainly affecting very close species. In Protocol v2, the correlation was 57.3 %, being greater in solid than in liquid media (71.7 % vs. 44.7 %, p < 0.05). Albeit not significant, a trend to a greater correlation for M. tuberculosis complex compared to non-tuberculous mycobacteria (NTM) (63.6 % vs. 55.5 %) was observed. In Protocol v3, the correlation was 73 %, with no significant differences between solid and liquid media (70.8 % vs. 75 %). In conclusion, MALDI-TOF MS may play a role in identifying mycobacterial species isolated from clinical samples, being faster than sequencing and hybridization-based techniques.

Aetiology of traveller's diarrhoea: evaluation of a multiplex PCR tool to detect different enteropathogens.

Traveller's diarrhoea (TD) is the most common illness reported in international travellers. TD is caused by a wide range of pathogens, including bacteria, viruses and parasites. Multiplex PCR assays can be especially useful for studying the aetiology of TD. The first objective of this study was to evaluate the utility of the commercially available multiplex PCR (xTAG(®) Gastrointestinal Pathogen Panel (GPP)) for the diagnosis of TD. A total of 185 stool specimens obtained from 174 patients were processed using the GPP assay. This test detected 86 pathogens in 67 stool samples (67/185, 36.2%). Sixteen pathogens out of 86 were also detected by routine testing. The remaining pathogens (n = 70) required further confirmation by alternative techniques. Finally, 60 out of 70 pathogens were confirmed. The second objective of this study was to analyse the aetiology of TD based on the results obtained by the GPP test and routine methods. The primary pathogens causing TD were Shigella (24.2%) followed by enterotoxigenic Escherichia coli (ETEC) (23.2%), enteroaggregative E. coli (14.7%) and Giardia (13.7%). Significant regional differences were observed for ETEC with 19.4% of TD cases acquired in Africa, 11.3% in Asia and none in South Central (SC) America (p 0.01), Giardia was found in 1.5% of cases among those who had travelled to Africa, 14.1% of those who had travelled to Asia and 3% of those who had travelled to SC America (p 0.01). In conclusion, the GPP test improved the detection of enteropathogens and allowed better assessment of the aetiology of TD.

[Gastrocolocutaneous fistula: an uncommon complication of percutaneous endoscopic gastrostomy]. / Fístula gastrocolocutánea: una infrecuente complicación de la gastrostomía endoscópica percutánea.

Endoscopic percutaneous gastrostomy is a safe technique although with potential complications before which the clinician has to be on alert in order to early detect them even after a long period of normal functioning. Most of them represent minor problems. Gastrocolocutaneous fistula is a rare but severe complication favored by some risk factors such as previous post-surgical adherences, deformities of the spine, or excessive gastric inflation at the time of performing the technique. We present the case of a patient with PEG with this complication that occurred after the first tube replacement. Our goal was in two senses: on the one hand, to analyze the preventive aspects and basic guidelines for a safe PEG placement to minimize the risks; on the other hand, to alert on the possible presence of this entity to prevent a progressive nutritional impairment. This complication ought to be included in the differential diagnosis of the diarrhea syndrome in the patient carrying a PEG. The diagnostic techniques of choice are radiologic tests such as CT scan and contrast media administration through the tube. Surgical therapy should be reserved to patients with acute peritonitis in order to perform a new gastrostomy.

Humoral and cellular immune responses of atopic individuals in a tropical environment.

In this study we compared immediate, intermediate and delayed skin test reactivity, total and specific serum IgE, IgG, A, M antibody and lymphocyte proliferative responses, between atopic and normal individuals in the tropical environment of Caracas, Venezuela (Lat. 10 degrees N). The allergenic extracts tested were prepared from house dust, mixed moulds and Aspergillus fumigatus. In lymphocyte stimulation the mitogen Concanavalin A was also employed, the cultures being supplemented with either autologous plasma, or a gamma globulin-depleted pool. The results revealed the association of immediate skin reactions with IgE antibody against house dust, and intermediate reactions with IgG, A, M antibody against moulds. No relation was, however, detected between delayed skin reactivity and in vitro lymphocyte transformation; skin reactions occurred at the highest frequency with moulds, while house dust provoked the strongest in vitro stimulation. Also, although the areas of positive delayed reactions were greatest in the atopic individuals, the lymphocyte proliferative responses were clearly highest in the normal subjects. The significance of the positivity of lymphocyte transformation tests in all of the study group, but lower reactivity in the atopics, is considered.

Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death.

Previously, we and others showed that signals relayed through the murine T cell Ag 4-1BB enhance primary T cell responses, and that blocking the interaction of 4-1BB with its ligand results in decreased responses to polyclonal activators and to alloantigens. Because 4-1BB expression is induced following primary stimulation, we investigated the role of signaling through this molecule in the reactivation of proliferating T cells. To this end, preactivated, 4-1BB-expressing T cells were restimulated in the presence of plate-immobilized mAbs directed against 4-1BB or the prototypic costimulatory molecule CD28. In this work, we show that in the presence of either signal, T cells respond to TCR cross-linking with strong proliferative responses and cytokine production; moreover, our findings indicate that T cell proliferation partially correlates with surface 4-1BB expression. In addition, our results suggest that Ab-mediated costimulatory signals can act independently of potential accessory B7-CD28/CTLA-4 (cytotoxic T lymphocyte Ag-4) interactions. Importantly, the characteristic DNA fragmentation and apoptotic cell death observed after TCR re-engagement are inhibited comparably in the presence of either 4-1BB or CD28 signaling.

Interrelationships between immediate, intermediate, and delayed cutaneous reactions and their in vitro correlates.

The mutual correlations between immediate, intermediate, and delayed cutaneous reactions, IgE, IgG, or combined IgG, A, and M antibody levels, and antigen- or mitogen-induced lymphocyte transformation were evaluated in a mixed group of allergic or non-allergic individuals. As might be expected, immediate and delayed reactions correlated significantly with IgE antibody levels and lymphocyte transformation, respectively. Intermediate time-course reactions did not correlate with IgG or IgG, A, and M antibody levels, but did so with immediate reactions, thus suggesting their "late phase" nature. Of particular interest was the finding of correlations that do not conform to the classical concept of the mechanisms involved in the generation of the different cutaneous reactions. Significant correlations were found between immediate or intermediate reactions and antigen-induced lymphocyte transformation, and between immediate and delayed reactions. These results are discussed in relation to recent suggestions that factors released from sensitized T cells can mediate early time-course reactions, and that such reactions may contribute to the manifestation of delayed-type hypersensitivity.

Potential role of 4-1BB in T cell activation. Comparison with the costimulatory molecule CD28.

The expression of the murine T cell Ag 4-1BB, a member of the TNF-R family, is induced by T cell activation. Previously, we and others had shown that signaling through 4-1BB enhanced proliferative T cell responses. To investigate a potential role for the interaction of 4-1BB with its ligand (4-1BBL) in T cell activation, we studied the ability of a soluble chimera of 4-1BB (4-1BBFc) to interfere with proliferative responses and cytokine production in models of activation dependent in intercellular interactions. The potential blocking effect of 4-1BBFc was compared with that of the chimeric molecule CTLA-4Ig, a reagent known to interfere with the interaction of CD28 (and/or CTLA-4) with B7 costimulatory receptors. In this study, we report that 4-1BBFc partially blocked both the activation of unfractionated splenocytes triggered by soluble anti-CD3 (anti-CD3s), and the more physiologically relevant responses to alloantigen. In addition, we show that both chimeric molecules partially blocked proliferative responses and IL-2 secretion by highly purified resting T cells activated with anti-CD3s in the presence of fixed accessory cells that express B7 receptors and 4-1BBL. Furthermore, in this model system, the blocking capacity of 4-1BBFc and CTLA-4Ig appears to correlate with the relative expression of their respective cognate receptors (4-1BBL and B7) on the accessory cell. Simultaneous addition of both blocking reagents produced an additive effect in the model systems studied.