Molecular Diagnosis of Toxoplasmosis in Immunocompromised Patients: a 3-Year Multicenter Retrospective Study

Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definitive diagnosis relies on parasite DNA detection, but little is known about the incidence and burden of disease in HIV-negative patients. A 3-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, in order to record the frequency of Toxoplasma gondii DNA detection in ICPs and to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, of 31,640 PCRs performed on samples from ICPs, 610 were positive (323 patients). Blood (n = 337 samples), cerebrospinal fluid (n = 101 samples), and aqueous humor (n = 100 samples) were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV⁺; the remaining 62% consisted of 72 HSCT, 14 solid organ transplant, and 26 miscellaneous immunodeficiency patients. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR result, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and they received specific treatment. Overall survival of allo-HSCT patients at 2 months was better in centers with PCR follow-up than in other centers (P < 0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide preemptive treatment and improve outcome.     

BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling

Activin A, BMP2, and BMP4, 3 members of the transforming growth factor-beta family, are involved in the regulation of hematopoiesis. Here, we explored the role of these molecules in human megakaryopoiesis using an in vitro serum-free assay. Our results highlight for the first time that, in the absence of thrombopoietin, BMP4 is able to induce CD34(+) progenitor differentiation into megakaryocytes through all stages. Although we have previously shown that activin A and BMP2 are involved in erythropoietic commitment, these molecules have no effect on human megakaryopoietic engagement and differentiation. Using signaling pathway-specific inhibitors, we show that BMP4, like thrombopoietin, exerts its effects on human megakaryopoiesis through the JAK/STAT and mTor pathways. Inhibition of the BMP signaling pathway with blocking antibodies, natural soluble inhibitors (FLRG or follistatin), or soluble BMP receptors reveals that thrombopoietin uses the BMP4 pathway to induce megakaryopoiesis, whereas the inverse is not occurring. Finally, we show that thrombopoietin up-regulates the BMP4 autocrine loop in megakaryocytic progenitors by inducing their production of BMP4 and up-regulating BMP receptor expression. In summary, this work indicates that BMP4 plays an important role in the control of human megakaryopoiesis.     

Multiple Influenza A (H3N2) Mutations Conferring Resistance to Neuraminidase Inhibitors in a Bone Marrow Transplant Recipient

Immunocompromised patients are predisposed to infections caused by influenza virus. Influenza virus may produce considerable morbidity, including protracted illness and prolonged viral shedding in these patients, thus prompting higher doses and prolonged courses of antiviral therapy. This approach may promote the emergence of resistant strains. Characterization of neuraminidase (NA) inhibitor (NAI)-resistant strains of influenza A virus is essential for documenting causes of resistance. In this study, using quantitative real-time PCR along with conventional Sanger sequencing, we identified an NAI-resistant strain of influenza A (H3N2) virus in an immunocompromised patient. In-depth analysis by deep gene sequencing revealed that various known markers of antiviral resistance, including transient R292K and Q136K substitutions and a sustained E119K (N2 numbering) substitution in the NA protein emerged during prolonged antiviral therapy. In addition, a combination of a 4-amino-acid deletion at residues 245 to 248 (Δ245-248) accompanied by the E119V substitution occurred, causing resistance to or reduced inhibition by NAIs (oseltamivir, zanamivir, and peramivir). Resistant variants within a pool of viral quasispecies arose during combined antiviral treatment. More research is needed to understand the interplay of drug resistance mutations, viral fitness, and transmission.     

Simultaneous presence in dogs of 2 zymodemes of the Leishmania infantum complex

The concomitant presence of two zymodemes of the leishmania infantum complex. MON-1 and MON-77, is reported in a dog with diffuse leishmaniasis. The zymodemes were present in both the skin and lymph nodes. Possible explanations for the presence of the two zymodemes are discussed.     

Quantitative dynamic cardiac 82Rb PET using generalized factor and compartment analyses.

We have addressed 2 major challenges of (82)Rb cardiac PET, noninvasive estimation of an accurate input function and absolute quantitation of myocardial perfusion, using a generalized form of least-squares factor analysis of dynamic sequences (GFADS) and a novel compartment analysis approach. METHODS: Left and right ventricular (LV + RV) time-activity curves (TACs) were generated from 10 rest/stress studies, and 30 myocardial TACs were modeled to cover a range of clinical values. Two-dimensional PET Monte Carlo simulations of the LV, RV, myocardium, and other organs were generated separately and combined using the above TACs to form 30 realistic dynamic (82)Rb studies. LV and RV TACs were estimated by GFADS and used as input to a 2-compartment kinetic analysis that estimates parametric maps of myocardial tissue extraction (k(1)) and egress (k(2)), as well as LV + RV contributions (f(v), r(v)), by orthogonal voxel grouping. In addition, 13 patients were injected with 2.22 +/- 0.19 GBq (60 +/- 5 mCi) of (82)Rb and imaged dynamically for 6 min at rest and during dipyridamole stress. RESULTS: In Monte Carlo simulations, GFADS yielded estimates of the 3 factors and corresponding factor images, with average errors of -4.2% +/- 6.3%, 3.5% +/- 4.3%, and 2.0% +/- 5.5% in the LV, RV, and myocardial factor estimates, respectively. The estimates were significantly more accurate and robust to noise than those obtained using TACs based on manually drawn volumes of interest (P < 0.01). The 2-compartment approach yielded accurate k(1), k(2), f(v), and r(v) parametric maps; the average error of estimates of k(1) was 6.8% +/- 3.6%. In all patient studies, our approach yielded robust estimates of k(1), k(2), f(v), and r(v), which correlated very well with the status of the subject and the catheterization results. CONCLUSION: Quantitative dynamic (82)Rb PET using generalized factor analysis of dynamic sequences and compartmental modeling yields estimates of parameters of absolute myocardial perfusion and kinetics with errors of <9%.