Incidence of tuberculous and non-tuberculous mycobacteria,differentiated by multiplex PCR,in clinical specimens of a large general hospital

OBJECTIVE:

To determine the incidence of Mycobacterium tuberculosis complex and non-tuberculous mycobacterial isolates in the routine setting of a large general hospital using an "in-house" multiplex polymerase chain reaction method and to establish a paradigm for the definitive identification of mycobacteria isolated using semi-automated equipment.

METHODS:

Established tests, including polymerase chain reaction restriction enzyme analysis, PNB, and NAP inhibition tests as the gold standard, showed 100% agreement with an IS6110/hsp65 multiplex polymerase chain reaction when used to identify stock strains (n = 117).

RESULTS:

In a subsequent study, 8,790 clinical specimens producing 476 isolates were evaluated with multiplex PCR and also showed 100% agreement in identification using PRA-polymerase chain reaction as the gold standard. The application of this technique to routine analysis was demonstrated in this study. A method was established with the initial application of multiplex PCR for all positive liquid cultures and the subsequent identification of non-tuberculous mycobacteria by polymerase chain reaction restriction enzyme analysis. In total, 77% of isolates belonged to the Mycobacterium tuberculosis complex, and 23% were non-tuberculous mycobacteria.

CONCLUSIONS:

Several non-tuberculous mycobacterial species were identified, primarily M. avium, but other potentially pathogenic species were also frequently observed, including M. fortuitum, M. abscessus, and M. kansasii. The expeditious communication of these data to the clinical staff was fundamental for the diagnosis of clinical cases. Even in settings where tuberculosis is of major importance, the incidence of non-tuberculous mycobacteria infection is substantial.     

Limitations of IL-2 and Rapamycin in Immunotherapy of Type 1 Diabetes

Administration of low-dose interleukin-2 (IL-2) alone or combined with rapamycin (RAPA) prevents hyperglycemia in NOD mice. Also, low-dose IL-2 cures recent-onset type 1 diabetes (T1D) in NOD mice, partially by boosting pancreatic regulatory T cells (T_reg cells). These approaches are currently being evaluated in humans. Our objective was to study the effect of higher IL-2 doses (250,000–500,000 IU daily) as well as low-dose IL-2 (25,000 IU daily) and RAPA (1 mg/kg daily) (RAPA/IL-2) combination. We show that, despite further boosting of T_reg cells, high doses of IL-2 rapidly precipitated T1D in prediabetic female and male mice and increased myeloid cells in the pancreas. Also, we observed that RAPA counteracted IL-2 effects on T_reg cells, failed to control IL-2–boosted NK cells, and broke IL-2–induced tolerance in a reversible way. Notably, the RAPA/IL-2 combination failure to cure T1D was associated with an unexpected deleterious effect on glucose homeostasis at multiple levels, including β-cell division, glucose tolerance, and liver glucose metabolism. Our data help to understand the therapeutic limitations of IL-2 alone or RAPA/IL-2 combination and could lead to the design of improved therapies for T1D.In type 1 diabetes (T1D), the immune system destroys the pancreatic β-cells (1). At clinical onset, ∼30% of β-cells are still able to produce insulin (2), thus stopping autoimmune destruction, which at this stage is a promising approach (3). Along the same lines, there is a growing list of phase I/II clinical trials based on immunomodulation that are currently being conducted in T1D patients (4).NOD mice, which develop spontaneous T1D, represent an accepted model for testing new therapies (5), the gold standard being that treatments that cure overt hyperglycemia in these mice may be most appropriate for translation into the clinic, as was the case for anti-CD3 antibodies (Abs) (6), which have been tested in patients with promising results (7). In addition, results from our own group showing that low-dose interleukin-2 (IL-2) can prevent (8) and revert disease in NOD mice (9) have led to the translation of this strategy into clinical trials in T1D patients (clinical trial reg. no. {"type":"clinical-trial","attrs":{"text":"NCT01353833","term_id":"NCT01353833"}}NCT01353833, clinicaltrials.gov).We have shown that in NOD mice, administration of low-dose IL-2 for 5 days induced the remission of new-onset T1D by specifically boosting regulatory T cells (T_reg cells) in the pancreas without activating pathogenic effector T cells (T_eff cells). However, remission was obtained in only 60% of treated mice, and half of them became diabetic again during the following months (9). Consequently, improving IL-2 therapy by optimizing dosing or combining IL-2 with other immunomodulatory drugs, such as rapamycin (RAPA), could be of great importance for the goal of translating this therapy to humans.RAPA has been used in clinical transplantation for many years (10), and it has been safely administered to T1D patients during islet transplantation (11,12). In mice, RAPA monotherapy can prevent T1D development (13); however, it is unable to induce disease reversal (14). Moreover, RAPA and IL-2 were found to be synergistic for the prevention of diabetes in NOD mice (13). Consequently, we decided to test whether RAPA could synergize with short-term IL-2 therapy to reverse T1D and reinforce the development of long-term tolerance.In this work, we have further studied the mechanisms of action of IL-2 and RAPA alone or in combination in the NOD model of T1D.     

Blood leukocytes from benznidazole-treated indeterminate chagas disease patients display an overall type-1-modulated cytokine profile upon short-term in vitro stimulation with trypanosoma cruzi antigens

ABSTRACT: BACKGROUND: Benznidazole (Bz)-chemotherapy is recommended to prevent Chagas disease progression, despite its limited efficacy during chronic disease. However, the host mechanisms underlying these benefits still remain to be elucidated. METHODS: In this study, we have used short-term whole blood cultures to describe the cytokine profile of Bz-treated Indeterminate Chagas disease patients-(INDt) as compared to untreated patients-(IND). RESULTS: Our findings showed that IND presented increased levels of IL-10+neutrophils, IL-12+ and IL-10+monocytes and IFN-gamma +NK-cells. Moreover, IND showed slight increase of IL- 4+CD4+T-cells and enhanced levels of IL-10+CD8+T-cells and B-cells. Additional analysis of cytokine Low and High producers also highlighted the presence of High cytokine producers within IND, including IL-10 from CD4+ T-cells and IFN-gamma from CD8+ T-cells, as compared to NI. The Bz-treatment lead to an overall cytokine down-regulation in the innate and adaptive compartments, including low levels of IL-12+ and IL-10+neutrophils and monocytes, IFN-gamma +NK-cells, IL-12+, TNF-alpha +, IFN-gamma + and IL-5+CD4+T-cells and IL-10+B-cells, along with basal levels of cytokine-expressing CD8+T-cells in INDt as compared to IND. The in vitro antigen stimulation shifted the cytokine profile toward a type 1-modulated profile, with increased levels of IL-12+ and IL-10+ monocytes, IFN-gamma + and IL-4+NK-cells along with TNF- alpha + and IFN-gamma +CD8+T-cells. Analysis of Low and High cytokine producers, upon in vitro antigen stimulation, further confirm these data. CONCLUSION: Together, our findings showed that the Bz treatment of Indeterminate Chagas' disease patients shifts the cytokine patterns of peripheral blood monocytes, NK-cells and CD8+ Tcells towards a long-lasting Type-1-modulated profile that could be important to the maintenance of a non-deleterious immunological microenvironment.     

Evolution of the retinal function by flash-ERG in one child suffering from neuronal ceroid lipofuscinosis CLN2 treated with cerliponase alpha: case report

Documenta Ophthalmologica - Neuronal ceroid lipofuscinoses (CLN) are neurodegenerative disorders among the most frequent, inherited as an autosomal recessive trait. Affected patients can present...     

Increasing sexually transmitted infection rates in young men having sex with men in the Netherlands, 2006–2012

Background

Men having sex with men (MSM) remain the largest high-risk group involved in on-going transmission of sexually transmitted infections (STI), including HIV, in the Netherlands. As risk behaviour may change with age, it is important to explore potential heterogeneity in risks by age. To improve our understanding of this epidemic, we analysed the prevalence of and risk factors for selected STI in MSM attending STI clinics in the Netherlands by age group.

Methods

Analysis of data from the national STI surveillance system for the period 2006–2012. Selected STI were chlamydia, gonorrhoea, infectious syphilis and/or a new HIV infection. Logistic regression was used to identify factors associated with these selected STI and with overall STI positivity. Analyses were done separately for MSM aged younger than 25 years and MSM aged 25 years and older.

Results

In young MSM a significant increase in positivity rate was seen over time (p?<?0.01), mainly driven by increasing gonorrhoea diagnoses, while in MSM aged 25 and older a significant decrease was observed (p?<?0.01). In multivariate analyses for young MSM, those who were involved in commercial sex were at higher risk (OR: 1.5, 95% CI: 1.2-1.9). For MSM aged 25 years and older this was not the case. Having a previous negative HIV test was protective among older MSM compared to those not tested for HIV before (OR: 0.8, 95% CI: 0.8-0.8), but not among younger MSM.

Conclusions

MSM visiting STI clinics remain a high-risk group for STI infections and transmission, but are not a homogenous group. While in MSM aged older than 25 years, STI positivity rate is decreasing, positivity rate in young MSM increased over time. Therefore specific attention needs to be paid towards targeted counselling and reaching particular MSM sub-groups, taken into account different behavioural profiles.

     

IL-8 and its CXCR1 and CXCR2 receptors participate in the control of megakaryocytic proliferation, differentiation, and ploidy in myeloid metaplasia with myelofibrosis

Myeloproliferation, myelofibrosis, and neoangiogenesis are the 3 major intrinsic pathophysiologic features of myeloid metaplasia with myelofibrosis (MMM). The myeloproliferation is characterized by an increased number of circulating CD34+ progenitors with the prominent amplification of dystrophic megakaryocytic (MK) cells and myeloid metaplasia in the spleen and liver. The various biologic activities of interleukin 8 (IL-8) in hematopoietic progenitor proliferation and mobilization as well as in neoangiogenesis prompted us to analyze its potential role in MMM. We showed that the level of IL-8 chemokine is significantly increased in the serum of patients and that various hematopoietic cells, including platelets, participate in its production. In vitro inhibition of autocrine IL-8 expressed by CD34+ cells with either a neutralizing or an antisense anti-IL-8 treatment increases the proliferation of MMM CD34(+)-derived cells and stimulates their MK differentiation. Moreover, addition of neutralizing anti-IL-8 receptor (CXC chemokine receptor 1 [CXCR1] or 2 [CXCR2]) antibodies to MMM CD34+ cells cultured under MK liquid culture conditions increases the proliferation and differentiation of MMM CD41+ MK cells and restores their polyploidization. Our results suggest that IL-8 and its receptors participate in the altered MK growth that features MMM and open new therapeutic prospects for this still incurable disease.     

Graft-Versus-Host Disease and Graft-Versus-Leukemia Effect in Mice Grafted With Peripheral Newborn Blood

We previously used peripheral newborn blood (NBB) as a possible invivo experimental model for cord blood (CB) transplantation and showedthat B10.D2 NBB cells successfully reconstituted adult (DBA/2 × B10.D2)F1 mice without causing graft-versus-host disease (GVHD),probably because of their phenotypic and functional immaturity. Here weinvestigated the influence of T-cell maturation occurring in NBB cellsduring the early postbirth period on the degree of engraftment, theincidence of GVHD, and the graft-versus-leukemia (GVL) potential. Theseparameters were compared in recipients grafted with bone marrow (BM)cells. We observed an increased percentage of CD4⁺ matureT cells accompanied by the acquisition of proliferative responses tophytohemagglutinin (PHA) and to allogeneic cells of day-5 NBB cells.The capacity of day-2 NBB to engraft was moderately reduced andrecipients developing GVHD were occasionally observed after the graftof day-5 NBB cells. No GVL effect was evidenced regardless of the timeof postbirth blood collection. However, the GVL effect can be obtainedby the delayed infusion of donor mature T cells to recipients graftedwith day-0 NBB, without causing GVHD. In contrast, the same protocolapplied to mice grafted with BM cells induced GVHD mortality of allrecipients. Interleukin (IL)-10 but not IL-2 messenger RNA wasexpressed in NBB cells as opposed to BM cells. These findings suggestthat, in terms of GVHD incidence, delayed infusion of mature T cells aspost-transplant tumor immunotherapy would be more effective whenapplied after CB than after BM transplantation.     

Alternative donor hematopoietic stem cell transplantation for mature lymphoid malignancies after reduced-intensity conditioning regimen: similar outcomes with umbilical cord blood and unrelated donor peripheral blood

We have reported encouraging results of unrelated cord blood transplantation for patients with lymphoid malignancies. Whether those outcomes are comparable to matched unrelated donor transplants remains to be defined. We studied 645 adult patients with mature lymphoid malignancies who received an allogeneic unrelated donor transplant using umbilical cord blood (n=104) or mobilized peripheral blood stem cells (n=541) after a reduced-intensity conditioning regimen. Unrelated cord blood recipients had more refractory disease. Median follow-up time was 30 months. Neutrophil engraftment (81% vs. 97%, respectively; P<0.0001) and chronic graft-versus-host disease (26% vs. 52%; P=0.0005) were less frequent after unrelated cord blood than after matched unrelated donor, whereas no differences were observed in grade II–IV acute graft-versus-host disease (29% vs. 32%), non-relapse mortality (29% vs. 28%), and relapse or progression (28% vs. 35%) at 36 months. There were also no significant differences in 2-year progression-free survival (43% vs. 58%, respectively) and overall survival (36% vs. 51%) at 36 months. In a multivariate analysis, no differences were observed in the outcomes between the two stem cell sources except for a higher risk of neutrophil engraftment (hazard ratio=2.12; P<0.0001) and chronic graft-versus-host disease (hazard ratio 2.10; P=0.0002) after matched unrelated donor transplant. In conclusion, there was no difference in final outcomes after transplantation between umbilical cord blood and matched unrelated donor transplant. Umbilical cord blood is a valuable alternative for patients with lymphoid malignancies lacking an HLA-matched donor, being associated with lower risk of chronic graft-versus-host disease.     

Development of a whole cell pneumococcal vaccine: BPL inactivation,cGMP production,and stability

Pneumococcal infections impose a large burden of disease on the human population, mainly in developing countries, and the current pneumococcal vaccines offer serotype-specific protection, but do not cover all pathogenic strains, leaving populations vulnerable to disease caused by non-vaccine serotypes. The pneumococcal whole cell vaccine is a low-cost strategy based on non-capsular antigens common to all strains, inducing serotype-independent immunity. Therefore, we developed the process for the cGMP production of this cellular vaccine. Initially, three engineering runs and two cGMP runs were performed in 60-L bioreactors, demonstrating the consistency of the production process, as evaluated by the growth curves, glucose consumption and metabolite formation (lactate and acetate). Cell recovery by tangential filtration was 92 ± 13%. We optimized the conditions for beta-propiolactone (BPL) inactivation of the bacterial suspensions, establishing a maximum cell density of OD₆₀₀ between 27 and 30, with a BPL concentration of 1:4000 (v/v) at 150 rpm and 4 °C for 30 h. BPL was hydrolyzed by heating for 2 h at 37 °C. The criteria and methods for quality control were defined using the engineering runs and the cGMP Lots passed all specifications. cGMP vaccine Lots displayed high potency, inducing between 80 and 90% survival in immunized mice when challenged with virulent pneumococci. Sera from mice immunized with the cGMP Lots recognized several pneumococcal proteins in the extract of encapsulated strains by Western blot. The cGMP whole cell antigen bulk and whole cell vaccine product lots were shown to be stable for up to 12 and 18 months, respectively, based upon survival assays following i.p. challenge. Our results show the consistency and stability of the cGMP whole cell pneumococcal vaccine lots and demonstrate the feasibility of production in a developing country setting.