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Background The detection of Trypanosoma cruzi genetic material in clinical samples is considered an important diagnostic tool for Chagas disease. We have previously demonstrated that PCR using clot samples yields greater sensitivity than either buffy coat or whole blood samples. However, phenol-chloroform DNA extraction from clot samples is difficult and toxic. The objective of the present study was to improve and develop a more sensitive method to recover parasite DNA from clot samples for the diagnosis of Chagas disease. Methodology/Principal findings A total of 265 match pair samples of whole blood–guanidine (GEB) and clot samples were analyzed; 150 were from Chagas seropositive subjects. DNA was extracted from both whole blood-guanidine samples, using a previously standardized methodology, and from clot samples, using a newly developed methodology based on a combination of the FastPrep technique and the standard method for GEB extraction. A qPCR targeting the nuclear satellite sequences was used to compare the sample source and the extraction method. Of the 150 samples from Chagas positive individuals by serology, 47 samples tested positive by qPCR with DNA extracted by both GEB and clot, but an additional 13 samples tested positive only in DNA extracted from clot. No serology-negative samples resulted positive when tested by qPCR. Conclusions The new methodology for DNA extraction from clot samples improves the molecular diagnosis of Chagas disease., Author summary Detection of nucleic acid has become an important tool for the diagnosis of Chagas disease. Whole blood samples are usually the source of DNA and qPCR the preferred technique to demonstrate the presence of T. cruzi DNA. Although DNA extracted from clot samples has shown higher sensitivity than from whole blood, DNA extraction is performed using phenol-chloroform, which has biohazard issues. We theorize that a clot traps parasites, making it a better source of DNA for Chagas diagnosis using PCR. The present study describes a new DNA extraction methodology from clot samples which avoids the use of phenol-chloroform. The new methodology was compared to the internationally standardized diagnostic method, which is based on extraction of DNA from whole blood preserved with guanidine EDTA and a commercial kit.

Ehrlichia spp. are emerging infectious pathogens, especially in the Americas. Although Ehrlichia canis is primarily a parasite of dogs, polymerase chain reaction-confirmed human infections have been reported from Mexico, Venezuela, and Costa Rica. This study reports the presence of E. canis DNA in 13.7% of 205 dogs from urban areas in Peru and of those, five were analyzed for phylogenetic variation using the Tandem Repeat Protein 36 (TRP36) gene. The use of the TRP36 gene for such analysis was validated against 16S rRNA and heat shock protein genes using Shannon’s entropy bioinformatic approach. When compared with other E. canis strains previously reported, three unique and novel E. canis strains were detected. In addition, the TRP36 amino acid tandem repeat sequences of the Peruvian strains share close similarity to an E. canis strain detected from four human blood bank samples in Costa Rica. This study reports for the first time domestic dogs infected with E. canis strains closely related to a zoonotic strain, which may be of public health concern as dogs can be chronically infected with this pathogen.

Ehrlichia spp. are emerging infectious pathogens, especially in the Americas. Although Ehrlichia canis is primarily a parasite of dogs, polymerase chain reaction-confirmed human infections have been reported from Mexico, Venezuela, and Costa Rica. This study reports the presence of E. canis DNA in 13.7% of 205 dogs from urban areas in Peru and of those, five were analyzed for phylogenetic variation using the Tandem Repeat Protein 36 (TRP36) gene. The use of the TRP36 gene for such analysis was validated against 16S rRNA and heat shock protein genes using Shannon's entropy bioinformatic approach. When compared with other E. canis strains previously reported, three unique and novel E. canis strains were detected. In addition, the TRP36 amino acid tandem repeat sequences of the Peruvian strains share close similarity to an E. canis strain detected from four human blood bank samples in Costa Rica. This study reports for the first time domestic dogs infected with E. canis strains closely related to a zoonotic strain, which may be of public health concern as dogs can be chronically infected with this pathogen.

Introduction. Gaucher disease (GD) is an infrequent progressive multisystem lysosomal storage disorder caused by the deficient activity of the lysosomal enzyme, glucocerebrosidase. A retrospective, single-center analysis of the clinical experience concerning the use of miglustat (N-butyldeoxynojirimycin), an oral inhibitor of glucosylceramide synthase, in type 1 Gaucher disease (GD1) was conducted to evaluate the efficacy, adverse events (AE), and outcome of miglustat therapy. Patients and methods. Six adult Caucasian patients with GD1 (two women and four men), aged 21–81 years (median age 59 years), were treated with miglustat between October 2005 and April 2011. All but one patient (83%) carried at least one allele with c.1226A>G (N370S) mutation in the GBA1 gene. Results. Weight loss, diarrhea, poor appetite, and tremor were frequently reported AE by the patients. All of them experienced at least 2 AE, and three patients (50%) experienced at least 4 AE. Only two out of six patients (33%) have used miglustat longer than 12 months, of which only one used it longer than 15 months. Conclusions. The major obstacle to successful miglustat therapy in GD1 was the high proportion of patients discontinuing their treatment due to the AE and the worsened quality of life. Further efforts are needed to improve tolerability of miglustat and, in consequence, compliance of patients treated with this orphan drug.

In this prospective randomized multicenter trial 93 patients, median age 72 years, with RAEB-t (n=25) and myelodysplastic syndrome (MDS)-AML (n=68) were allocated to a standard induction chemotherapy regimen (TAD 2+7) with or without addition of granulocyte-macrophage-CSF (GM-CSF). The overall complete remission (CR) rate was 43% with no difference between the arms. Median survival times for all patients, CR patients, and non-CR patients were 280, 550, and 100 days, respectively, with no difference between the arms. Response rates were significantly better in patients with serum lactate dehydrogenase (S-LDH) levels

High-dose cytarabine in upfront therapy for adult patients with acute lymphoblastic leukaemia

Bartonella species are emerging infectious organisms transmitted by arthropods capable of causing long-lasting infection in mammalian hosts. Among over 30 species described from four continents to date, 15 are known to infect humans, with eight of these capable of infecting dogs as well. B. bacilliformis is the only species described infecting humans in Peru; however, several other Bartonella species were detected in small mammals, bats, ticks, and fleas in that country. The objective of this study was to determine the serological and/or molecular prevalence of Bartonella species in asymptomatic dogs in Peru in order to indirectly evaluate the potential for human exposure to zoonotic Bartonella species. A convenient sample of 219 healthy dogs was obtained from five cities and three villages in Peru. EDTA-blood samples were collected from 205 dogs, whereas serum samples were available from 108 dogs. The EDTA-blood samples were screened by PCR followed by nucleotide sequencing for species identification. Antibodies against B. vinsonii berkhoffii and B. rochalimae were detected by IFA (cut-off of 1∶64). Bartonella DNA was detected in 21 of the 205 dogs (10%). Fifteen dogs were infected with B. rochalimae, while six dogs were infected with B. v. berkhoffii genotype III. Seropositivity for B. rochalimae was detected in 67 dogs (62%), and for B. v. berkhoffii in 43 (40%) of the 108 dogs. Reciprocal titers ≥1∶256 for B. rochalimae were detected in 19% of dogs, and for B. v. berkhoffii in 6.5% of dogs. This study identifies for the first time a population of dogs exposed to or infected with zoonotic Bartonella species, suggesting that domestic dogs may be the natural reservoir of these zoonotic organisms. Since dogs are epidemiological sentinels, Peruvian humans may be exposed to infections with B. rochalimae or B. v. berkhoffii., Author Summary Bartonella are bacteria transmitted by fleas, ticks, sandflies and other insects capable of infecting humans, domestic animals, livestock and wildlife, including marine mammals. In humans, they cause diseases such as trench fever, cat scratch disease, endocarditis, fever of unknown origin and have been recently associated with neurologic and neurocognitive abnormalities. Bartonella bacilliformis was first described in Peru in 1913, and it has never been detected in animals. Despite the fact that 14 other Bartonella species have been detected infecting humans around the world, no other Bartonella species has yet been described from Peruvian humans or domestic animals. We documented a significant number of healthy domestic dogs in Peru infected or exposed to two Bartonella species (B. rochalimae and B. vinsonii subsp. berkhoffii), which are known to cause disease in humans. These same species were previously detected in human fleas and dog ticks in Peru, suggesting that vector transmission between dogs and humans may be possible. While the role of dogs as a source of Bartonella species for direct transmission to humans is not well understood, preventive measures including vector control in dogs should be implemented to prevent human infection.

In acute lymphatic leukaemia (ALL) treated with allogenic stem cell transplantation limited chronic graft versus host disease (cGVHD) is associated with a higher 5 year disease free survival. This indicates an important graft-versus-leukemia (GVL) effect, although this effect is less pronounced in ALL than in other malignancies. B-cell depletion using Rituximab is a treatment option in therapy refractory cGVHD that has been used successfully in a limited number of patients with an approximate response rate of 70%. Only two ALL patients with cGVHD treated with Rituximab are found in the literature. Here we describe a pre-B-ALL patient with therapy refractory cGVHD who relapsed only one month after receiving Rituximab therapy for cGVHD. The Rituximab therapy may have triggered the relapse by targeting the shared cGVHD and GVL mechanisms, diminishing the GVL effect. Although no definite conclusions can be drawn from one case this might have implications for the use of Rituximab in cGVHD in ALL patients.

The International Prognostic Scoring System (IPSS), based on the number of cytopenias, percentage of bone marrow blasts and cytogenetics, is an important prognostic tool for patients with myelodysplastic syndrome (MDS). In addition, factors such as high bone marrow cellularity and lactate dehydrogenase levels have been associated with an adverse outcome, spontaneously and after chemotherapy. Recently, increased bone marrow angiogenesis, measured as, e.g. microvascular density (MVD), was reported to be more intense in high-risk than in low-risk MDS. To assess the prognostic role of MVD in MDS, a cohort of 56 patients, thoroughly investigated for various clinical and morphological parameters, were followed-up for survival > or =60 months after the diagnostic analysis. As a group MDS patients had higher MVD compared to healthy controls (p

The Image Understanding Architecture (IUA) effort is now entering a second phase. The IUA proof-of-concept prototype has been completed and our experience with both the hardware and extensive software simulations are guiding the development of a second generation of the IUA. Furthermore, the initial research-oriented software development environment is currently being replaced by a sophisticated set of application-oriented tools. Thus, the IUA effort is in the process of making the transition from an isolated research project to being in a position of accessibility to the wider community. This article describes the current status of the effort and some of our plans for the future. IUA development is taking place at three sites: the University of Massachusetts at Amherst, Hughes Research Laboratories in Malibu, and Amerinex Artificial Intelligence Inc. The article is thus divided into major sections that describe the efforts taking place at each site.

To investigate the origin of the cyclic changes in the rate of endogenous carbon-monoxide production (nCO) during the menstrual cycle, haem turnover was determined before and after chorion gonadotropic hormone-induced ovulation in six female rabbits. 14C-labelled delta-aminolevulinic acid and glycine were administered and the excretion rate of 14CO (A14CO) was measured for determination of hepatic and bone-marrow haem turnover, respectively. Carbon-monoxide production (nCO) was measured to estimate total haem turnover. After ovulation A14CO was increased significantly the first 2 h of the early labelled peak after 14C-ALA administration and was increased also at the first determination during the early peak after 14C-glycine but statistically not significantly. The total excretion of labelled CO during the period of the early labelled peak was not increased with any of these precursors for haem synthesis. On the other hand nCO was increased 34% (P less than 0.05) during the post-ovulation period. As the increase in 'unassigned' haem turnover was small and may be unaccompanied by a contemporary increase in bilirubin/CO production, it was concluded that the increase in nCO during the post-ovulation period essentially depends on increased destruction of circulating red cells in the rabbit.