Molecular detection of Bartonella in fleas (Hexapoda, Siphonaptera) collected from wild rodents (Cricetidae, Sigmodontinae) from Argentina.

Bartonella are facultative intracellular Gram-negative bacteria, transmitted mainly by hematophagous arthropods, and the rodents act as a natural reservoir. Different species of Bartonella associated with rodents have been implicated as causing human disease. Studies from Argentina are scarce and no Bartonella from fleas have been reported previously. The present study investigated the presence of Bartonella spp. in fleas associated with sigmodontine rodents in four localities of the Santa Cruz Province, Argentina. In total, 51 fleas (four species) were analysed of which 41.2% were found to be positive for the gltA gene fragment via a nested polymerase chain reaction. All positive fleas were of the species Neotyphloceras crackensis from three different localities. Eight of the 21 amplified samples were sequenced, and the presence of three different genotypes was detected with an identity of 95.5-98.8% amongst themselves. Bartonella genotypes from American rodents and rodent fleas were recovered in a monophyletic group. Similarly, most of the Peruvian and all Argentinean variants constitute a natural group sister of the American remainder. The importance of the Bartonella spp. with respect to public health is unknown, although future studies could provide evidence of the possible involvement of N. crackensis in the Bartonella transmission cycles.

[Molecular detection of Rickettsia massiliae and Anaplasma platys infecting Rhipicephalus sanguineus ticks and dogs, Bahía Blanca (Argentina)]. / Detección molecular de Rickettsia massiliae y Anaplasma platys en garrapatas Rhipicephalus sanguineus y caninos domésticos del municipio de Bahía Blanca (Argentina).

BACKGROUND: Rickettsioses, ehrlichioses and anaplasmoses are caused by Gram negative obligate intracellular bacteria and transmitted mainly by arthropods. AIM: To detect and perform the molecular characterization of these pathogens in ticks and domestic dogs in Bahia Blanca City (Buenos Aires, Argentina). METHODS: Fifty six blood samples from dogs and 82 ticks (75 Rhipicephalus sanguineus and 7 Amblyomma tigrinum) were studied. The samples were analyzed by PCR for Rickettsia (intergenic space 23S-5S rRNA), Ehrlichia/Anaplasma (16S rRNA), and Anaplasma platys (16S rRNA). RESULTS: 12% of R. sanguineus resulted positive for Rickettsia, identified by sequencing as Rickettsia massiliae; and 37.5% of the canine blood samples analyzed were positive for A. platys. Molecular characterization was also performed by amplification of the fragment of the citrate synthase gene (gltA) (Rickettsia genus) and the groESL gene (A. platys). Phylogenetic trees were constructed using the neighbor-joining method. These trees revealed that sequences obtained are similar to those from other geographical regions. CONCLUSION: The results indicate the presence of R. massiliae in R. sanguineus ticks for the second time in an urban area of South America and A. platys infection in dogs, being the southernmost region of Argentina where it has been notified.

Estimating the savings of a national project to prevent healthcare-associated infections in intensive care units.

BACKGROUND: Healthcare-associated infections (HAIs) have a significant impact on patients' morbidity and mortality, and have a detrimental financial impact on the healthcare system. Various strategies exist to prevent HAIs, but economic evaluations are needed to determine which are most appropriate. AIM: To present the financial impact of a nationwide project on HAI prevention in intensive care units (ICUs) using a quality improvement (QI) approach. METHODS: A health economic evaluation assessed the financial results of the QI initiative 'Saúde em Nossas Mãos' (SNM), implemented in Brazil between January 2018 and December 2020. Among 116 participating institutions, 13 (11.2%) fully reported the aggregate cost and stratified patients (with vs without HAIs) in the pre-intervention and post-intervention periods. Average cost (AC) was calculated for each analysed HAI: central-line-associated bloodstream infections (CLABSIs), ventilator-associated pneumonia (VAP) and catheter-associated urinary tract infections (CAUTIs). The absorption model and time-driven activity-based costing were used for cost estimations. The numbers of infections that the project could have prevented during its implementation were estimated to demonstrate the financial impact of the SNM initiative. RESULTS: The aggregated ACs calculated for each HAI from these 13 ICUs - US$8480 for CLABSIs, US$10,039 for VAP, and US$7464 for CAUTIs - were extrapolated to the total number of HAIs prevented by the project (1727 CLABSIs, 3797 VAP and 2150 CAUTIs). The overall savings of the SNM as of December 2020 were estimated at US$68.8 million, with an estimated return on investment (ROI) of 765%. CONCLUSION: Reporting accurate financial data on HAI prevention strategies is still challenging in Brazil. These results suggest that a national QI initiative to prevent HAIs in critical care settings is a feasible and value-based approach, reducing financial waste and yielding a significant ROI for the healthcare system.

Detection of Neorickettsia sp. in Oligoryzomys flavescens rodent from a protected urban area in Buenos Aires City (Argentina).

Rodents play an important role in vector-borne pathogens cycle. To detect Anaplasma, Ehrlichia, Neorickettsia, Rickettsia and Borrelia species in rodents from a protected urban area in Buenos Aires City (Argentina) were analyzed 203 organ pools of Mus musculus, Oligoryzomys flavescens, Rattus norvegicus, Deltamys kempi and Scapteromys aquaticus by PCR. Only one O. flavescens (1.2%) was positive by PCR for 16S rRNA fragment for the Anaplasmataceae family and the sequence had 99.7% identity with Neorickettsia risticii. Plus, the sequence obtained for a fragment of the p51 gene for the genus Neorickettsia from positive sample had 95.3-96.1% identity with N. risticii found previously in bats Tadarida brasiliensis from Buenos Aires City. Our study presents the first finding of Neorickettsia in rodents from natural environment, but further studies are necessary about these vector-borne bacteria and the rol of rodents in its epidemiology.

Two novel Ehrlichia strains detected in Amblyomma tigrinum ticks associated to dogs in peri-urban areas of Argentina.

The aim of this work was to describe two novel strains of Ehrlichia associated to Amblyomma tigrinum from Argentina. Molecular detection of agents belonging to the family Anaplasmataceae was performed targeting three different loci: 16S rRNA gene, dsb gene and a fragment of groESL heat shock operon. The results have shown that two different strains of Ehrlichia sp. associated to A. tigrinum are circulating in peri-urban areas of Argentina. The Ehrlichia strain detected in ticks from San Luis Province, named as Ehrlichia sp. strain San Luis, is closely related to the Ehrlichia chaffeensis. The novel Ehrlichia strain detected in Córdoba Province, named as Ehrlichia sp. strain Córdoba, is phylogenetically related to three Ehrlichia strains from Brazil, two of them isolated from wild carnivorous and the third one isolated from horse. Even though Ehrlichia sp. strain Córdoba was clustered with the three Ehrlichia strains from Brazil, the genetic similarity was too low to consider them as the same taxonomic entity. Blood samples of dogs were positive to Anaplasma platys. The association of these two novel strains with A. tigrinum has epidemiological relevance because adult stages of this tick species are common parasite of dogs in rural and peri-urban areas and they are aggressive to humans. The presence of these two novel Ehrlichia strains implies a potential epidemiological risk in Argentina because the species of the genus Ehrlichia are known to be pathogenic to both domestic mammals and humans.

Infection with Ehrlichia canis and Anaplasma platys (Rickettsiales: Anaplasmataceae) in two lineages of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) from Argentina.

Natural infection with Ehrlichia canis and Anaplasma platys in ticks belonging to the tropical and temperate lineages of Rhipicephalus sanguineus sensu lato from Argentina was evaluated. Samples were tested for Ehrlichia canis infection by PCR assays using 16S rRNA, dsb and p28 gene, while detection of A. platys was performed with 16S rRNA and groESL gene. The assignment of the ticks to each lineage was corroborated with 16S rDNA sequences. All ticks infected with E. canis and A. platys belonged to the tropical lineage. These results constitute the first record of E. canis infection in R. sanguineus s.l ticks from Argentina. No ticks from the temperate lineage were found to be infected with E. canis, coinciding with previous studies performed in Argentina and Uruguay where E. canis infection was not detected in R. sanguineus s.l from the temperate lineage. Because the presence of the tropical lineage of R. sanguineus s.l has been documented in tropical areas of northern Argentina between 22° and 24° of south latitude, the findings of this work indicate that transmission of E. canis and A. platys to dogs by R. sanguineus s.l probably occurs along this region.

Isolation of Rickettsia massiliae from Rhipicephalus sanguineus Ticks, Buenos Aires (Argentina).

Rickettsia massiliae , a member of the spotted fever group of Rickettsia, was first isolated from a Rhipicephalus turanicus tick in France. In the New World, it has been detected in Rhipicephalus sanguineus ticks from different geographical locations in Argentina and the United States, but it has only been isolated in Arizona. The aim of this study was the isolation and genetic characterization of R. massiliae from R. sanguineus ticks collected from dogs in Buenos Aires city, Argentina. In total, 49 R. sanguineus ticks were collected from 10 dogs and grouped into 10 pools of 4-5 specimens. With a PCR assay, which detects a fragment of the Rickettsia genus-specific 23S-5S intergenic space, 1 pool of 5 ticks was found positive. Generated sequences exhibited 100% identity with R. massiliae . A new isolate, named CABA, was obtained from this pool by inoculating it into monolayers of Vero cells. Genotypic characteristics were determined, and results showed that fragments of the 23S-5S intergenic space, ompA, ompB, gltA, htrA, and sca1 genes had great similarity with R. massiliae strain Bar29 (Spain). Although few human cases have been confirmed for this pathogen, its circulation in urban areas is of great importance to public health. This isolation improves knowledge of the circulating pathogen and could improve future diagnostic processes as it allows the production of more specific antigens for serological testing.

Rickettsia infection in Amblyomma tonelliae, a tick species from the Amblyomma cajennense complex.

The present study was performed to evaluate the Rickettsia infection in Amblyomma tonelliae ticks from Argentina. All ticks were subjected to DNA extraction and tested by a battery of PCRs to amplify fragments of four rickettsial genes, 23S-5S, gltA, ompA and htrA. Two ticks were positive. The Rickettsia detected in one tick represents a new lineage which is named Rickettsia sp. strain El Tunal. This new strain belongs to the canadensis group because it is closely related to Rickettsia monteiroi, Rickettsia canadensis and Candidatus "Rickettsia tarasevichiae". They clustered together on a high supported clade with both gltA and htrA genes. The other positive tick was infected with Candidatus "Rickettsia amblyommii". The results presented in this study constitute the first records of Rickettsia infection in A. tonelliae ticks. However, the medical relevance of these findings should be considered cautiously because the pathogenicity of Rickettsia sp. strain El Tunal and Candidatus "R. amblyommii" remains undetermined.

Liver alterations in antituberculosis regimens containing pyrazinamide.

A group of alcoholic tuberculous patients was given a pyrazinamide-containing regimen, and the liver was studied on admission and after two months of intensive treatment with IRSZ. Alterations in liver histology and in liver function tests found on admission improved or disappeared after two months' chemotherapy. A control group, give IRSE, showed similar results. A third group of nonalcoholic patients was given Z alone for 15 days, and liver tolerance was also excellent. These data support the conclusion that tuberculous alcoholic patients, in the absence of significant and persistent hepatic dysfunction, can be given Z-containing regimens. In all cases, a careful monitoring of hepatic function, with monthly SGPT and bilirubin determinations, is recommended.

In patients with COPD, treatment with a combination of formoterol and ipratropium is more effective than a combination of salbutamol and ipratropium : a 3-week, randomized, double-blind, within-patient, multicenter study.

STUDY OBJECTIVES: To compare the efficacy of adding formoterol or salbutamol to regular ipratropium bromide treatment in COPD patients whose conditions were suboptimally controlled with ipratropium bromide alone. DESIGN: A randomized, double-blind, double-dummy, two-period, crossover clinical trial. SETTING: Twenty-four clinics and university medical centers in nine countries. PATIENTS: One hundred seventy-two patients with baseline FEV(1) < or = 65% predicted, with FEV(1) reversibility to salbutamol not exceeding the normal variability of the measurement, and symptomatic despite regular treatment with ipratropium bromide. INTERVENTIONS: Each patient received two treatments in random order: either inhaled formoterol dry powder, 12 microg bid, in addition to ipratropium bromide, 40 microg qid for 3 weeks, followed by salbutamol, 200 microg qid, in addition to ipratropium, 40 microg qid for 3 weeks, or vice versa. MEASUREMENTS AND RESULTS: Efficacy end points included morning premedication peak expiratory flow (PEF) during the last week of treatment (primary end point), the area under the curve (AUC) for FEV(1) measured for 6 h after morning dose on the last day of treatment, and symptom scores (from daily diary recordings). Morning PEF and the AUC for FEV(1) were significantly better for formoterol/ipratropium than for salbutamol/ipratropium (p = 0.0003 and p < 0.0001, respectively). The formoterol/ipratropium combination also induced a greater improvement in mean total symptom scores (p = 0.0042). The safety profile of the two treatments was comparable. CONCLUSIONS: In COPD patients requiring combination bronchodilator treatment, the addition of formoterol to regular ipratropium treatment is more effective than the addition of salbutamol.

CD4+ T-lymphocytopenia in severe pulmonary tuberculosis without evidence of human immunodeficiency virus infection.

SETTING: A large public hospital in Buenos Aires, Argentina. OBJECTIVE: To determine the number of blood CD4 and CD8 T-lymphocytes in male human immunodeficiency virus (HIV) negative patients with severe pulmonary tuberculosis. DESIGN: Seventeen patients with severe pulmonary tuberculosis (SPT), with a mean age of 44.1 years, all HIV negative, had on admission lost 20% or more of their normal weight. Ten male HIV negative pulmonary tuberculosis patients (PT), with a mean age of 25.2 years, in good general condition, acted as a control group. Patients from both groups had a blood CD4/CD8 count before treatment. RESULTS: In the SPT patients, the CD4/CD8 count before treatment yielded a mean of 341.25 +/- 142.73/ mm3 for CD4 and 259.33 +/- 100.89/mm3 for CD8. Three patients died a few weeks after starting treatment; on admission they had 180,220 and 280 CD4/ mm3, respectively. Patients in good general condition yielded 721.40 +/- 272.20 for CD4 (P < 0.01, t = 4.216) and 416.67 for CD8. At the same time, five normal volunteers, with a mean age of 35.60 +/- 10.45 years, had mean CD4 and CD8 counts of 906 +/- 75.37 and 360 +/- 190.79, respectively. CONCLUSION: Based on the findings of this study, we feel that it is of value to measure the CD4 and CD8 T-lymphocyte counts in STP patients with a compromised general condition and with significant weight loss at the beginning of treatment. Those patients with a CD4 count of < 300/mm3 have a very poor prognosis and, in addition to the regular antituberculosis drugs, will require intensive care during the first weeks of treatment.

[Physiopathology, diagnosis and treatment of severe chronic hypoxemia. Role of residential chronic oxygen therapy]. / Fisiopatología, diagnostico y tratamiento de la hipoxemia crónica grave. Rol de la oxigenoterapia crónica domiciliaria.

The purpose of this article is to review the etiological and pathophysiological aspects of chronic severe hypoxemia (CSH) and to determine the indications of long-term oxygen therapy (LTOT). Three hypothesis are presented and analyzed: 1) CSH is harmful to the economy; 2) LTOT is therefore useful; 3) LTOT is not toxic and does not imply major risks than the benefits that it offers. Changes are produced by prolonged exposure to low levels of O2 leading to a sustained increase in pulmonary artery pressure. Secondary pulmonary hypertension (SPH) due to chronic hypoxemia is much more subtle and less symptomatic than that produced by other pathologies. Chronic obstructive pulmonary disease is the most common cause of CSH; these patients have a poor prognosis associated to the hypoxemia and its effects, being a PaO2 below 60 mmHg one of the most precise factors of mortality. Patients selection criteria for LTOT different sources for home oxygen therapy, methods of administration and finally an update of LTOT situation in our country and abroad are discussed.

[Pharmacokinetic interaction of ketoconazole, isoniazid and rifampicin]. / Interacción farmacocinética entre ketoconazol, isoniacida y rifampicina.

Eight male tuberculous patients, between 20 and 60 years of age, were given Isoniazid 5 mg/kg and Ketoconazole 200 mg, first one at a time and then associated. Plasma concentrations were measured 0, 2 and 5 hs after taking the drugs. Isoniazid was measured by spectrophotometry and Ketoconazole by the microbiologic method with Candida albicans as test microorganism. When both drugs were given simultaneously Ketoconazole plasma concentration decreased 75% at 2 hs (p less than 0.025) and 85% at 5 hs (p less than 0.05), whereas that of Isoniazid remained unchanged (Table 1). Mean half-life of Isoniazid was 3.9 +/- 1.4 hs in 7 slow acetylators and 1.1 hs in one fast acetylator when given one at a time and 4.4 +/- 1.5 hs when given simultaneously. A similar study was conducted on 11 tuberculous patients who were given Rifampicin 10 mg/kg and Ketoconazole 200 mg, one at a time and concurrently. Rifampicin was measured by high pressure liquid chromatography. When Rifampicin and Ketoconazole were given concurrently plasma concentration of both drugs was reduced: Ketoconazole decreased 85% at 2 hs (p less than 0.025) and 98% at 5 hs (p less than 0.025) whereas Rifampicin decreased 45% at 2 hs (p less than 0.005) and 40% at 5 hs (p less than 0.005) (Table 2). Mean half-life of Rifampicin was 3.5 +/- 0.8 and 4.2 +/- 1.1 hs, respectively, when it was given alone and concurrently. Studies on chemical interactions between Isoniazid and Ketoconazole and between Rifampicin and Ketoconazole yielded negative results.(ABSTRACT TRUNCATED AT 250 WORDS)

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Non-inferiority trial between two dry-powder inhalers containing fluticasone/salmeterol in asthmatic patients.

Asthma is an increasing pathology with poor compliance. Achievement of control is possible but under intensive treatment. In this setting, fluticasone/salmeterol association delivered by dry powder inhalers is a valuable and proved option. A prospective, parallel, open-label, phase IV, multicentre non-inferiority study was conducted to determine therapeutic similarity between 2 different inhalers: Generic DPI and Diskus®, which both deliver a fluticasone/salmeterol association (CAS 80474-14-2/CAS 89365-50-4). A 103 uncontrolled asthmatic patients were randomly assigned in 2 groups, Generic (G) and Diskus® (D), and received the association for 18 weeks through the appropriate device. They were evaluated according to Asthma Quality of Life Questionnaire and GINA/NIH guidelines. To demonstrate non-inferiority, the estimation of the Relative Risk between the Global Score Rate per group with its 95% confidence interval was calculated and compared against a non-inferiority margin obtained from a previous study. The Global Score Rate was 82% for G Group and 83% for D Group. The RR was 1.0124 (95% CI: 0.847-1.210). The margin set at 0.832 was not reached by the lower 95% CI (z=-2.097; p=0.018) pointing out non-inferiority. The results have demonstrated non-inferiority between groups. Thus, the 2 products are therapeutically similar.

Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21(Cip1) expression and hampers tumour cell growth in vitro and in vivo.

Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21(Cip1) level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS.

Efficacy and safety of ipratropium bromide/albuterol delivered via Respimat inhaler versus MDI.

We compared the efficacy and safety of ipratropium bromide/albuterol delivered via Respimat inhaler, a novel propellant-free inhaler, versus chlorofluorocarbon (CFC)-metered dose inhaler (MDI) and ipratropium Respimat inhaler in patients with COPD. This was a multinational, randomized, double-blind, double-dummy, 12-week, parallel-group, active-controlled study. Patients with moderate to severe COPD were randomized to ipratropium bromide/albuterol (20/100mcg) Respimat inhaler, ipratropium bromide/albuterol MDI [36mcg/206mcg (Combivent Inhalation Aerosol MDI)], or ipratropium bromide (20mcg) Respimat inhaler. Each medication was administered four times daily. Serial spirometry was performed over 6h (0.15min, then hourly) on 4 test days. The primary efficacy variable was forced expiratory volume in 1s (FEV(1)) change from test day baseline at 12 weeks. A total of 1209 of 1480 randomized, treated patients completed the study; the majority were male (65%) with a mean age of 64 yrs and a mean screening pre-bronchodilator FEV(1) (percent predicted) of 41%. Ipratropium bromide/albuterol Respimat inhaler had comparable efficacy to ipratropium bromide/albuterol MDI for FEV(1) area under the curve at 0-6h (AUC(0-6)), superior efficacy to ipratropium Respimat inhaler for FEV(1) AUC(0-4) and comparable efficacy to ipratropium Respimat inhaler for FEV(1) AUC(4-6). All active treatments were well tolerated. This study demonstrates that ipratropium bromide/albuterol 20/100mcg inhaler administered four times daily for 12 weeks had equivalent bronchodilator efficacy and comparable safety to ipratropium bromide/albuterol 36mcg/206mcg MDI, and significantly improved lung function compared with the mono-component ipratropium bromide 20 mcg Respimat inhaler. [Clinical Trial Identifier Number: NCT00400153].