Evaluation of B. melitensis whole-cell lysate antigen-based indirect ELISA for the serodiagnosis of caprine brucellosis.

Different fractions of Brucella (B) abortus or Brucella melitensis have been used as antigens for the detection of anti-Brucella antibodies in goat sera, being their accomplishment cumbersome and time consuming. In an attempt to achieve a simpler enzyme-linked immunosorbent assay (ELISA) antigen preparation method for serodiagnosis of caprine brucellosis, we developed and evaluated a B. melitensis whole-cell lysate antigen-based indirect ELISA (Bm-WCL iELISA). A total of 162 serum samples from female crossbred goats collected from non-vaccinated herds against brucellosis were classified according to the buffered plate antigen (BPA) screening test and the complement fixation (CF) test and used for the indirect ELISA (iELISA) evaluation. The Bm-WCL iELISA showed a high Se and Sp [95.7% (CI 88.1% - 98.8%), and 92.4% (CI 83.4% - 96.7%), respectively] to detect the serological response against Brucella in commercial goat herds, and an almost perfect agreement with combined official tests results (κ = 0.88), when goat sera with concordant results in both official serological tests (BPA and CF; n = 136) were used. However, the agreement dropped to substantial (k > 0.73) when 26 goat serum samples with BPA and CF not concordant results were incorporated for the iELISA performance evaluation and the comparison was made for each test independently. Comparison of the Bm-WCL iELISA results with Brucella abortus sLPS iELISA showed almost perfect agreement (κ > 0.83). Even when a larger number of samples are needed to validate this test, these preliminary results encourage the optimization of the Brucella melitensis whole cell lysate antigen-based iELISA.

Two Synchronous Neonatal Tumors: An Extremely Rare Case.

We report a case of a newborn with two synchronous tumors-sialoblastoma and hepatoblastoma-diagnosed at 20 weeks of gestation by magnetic resonance imaging (MRI) and ultrasonography (US). The aim of this study was to describe the management of this case together with a review of the literature. Our patient had a large facial tumor associated with extremely high alpha-fetoprotein levels. Diagnosis of the tumors was made by surgical biopsy, showing typical features in both. Sialoblastoma is a potentially aggressive tumor. In our case, the Ki67 index in the sialoblastoma was between 20 and 30%, indicating a possibly unfavorable behavior. The infant underwent surgery and chemotherapy in different steps. Complete surgical resection with clean margins is considered to be the best treatment option for sialoblastoma. Only four similar cases were previously reported. Timely management by a multidisciplinary team is essential in these difficult cases. In our patient, outcome was good at the time of this report.

Clinical characteristics of vulnerable populations hospitalized and diagnosed with COVID-19 in Buenos Aires, Argentina.

There is not in Argentina publications regarding the presentation of patients with COVID-19 requiring hospitalized and emergency care in vulnerable populations (lower incomes and less education tend at greater risk for poor health status and healthcare access), and it has few reports in developing countries. The objective is to determine whether in the care of vulnerable patients, to succeed against COVID-19, multiple public health tools and interventions will be needed to minimize morbidity and mortality. The study is a prospective cohort investigation of patients with lab-confirmed COVID-19, who required to any of the Health Centers response from April 8, 2020, to August 18, 2020. In Buenos Aires Metropolitan Area (AMBA), April 8, 2020 the virus was identified in patients hospitalized in the "Southeast Network" (SN), AMBA. SN covering an area of 661 square kilometers, with 1.8 million inhabitants residing in urban, and rural areas. A total of 14 health centers with different levels of care complexity provide care to patients in the region. The information of each patient with COVID-19 evaluated by SN, was incorporated in an Epidemiological Dashboard. The investigation was designed and reported with consideration of observational studies in epidemiology. We describe the hospitals presentation and care of persons who required SN response and were ultimately diagnosed with COVID-19. From April 8, 2020, to August 18, 2020, were included 1495 patients with lab-confirmed COVID-19 in SN. A total of 58% patients were men, and the mean age (SD) was 48.9 (15.59) years. Eighty one percent patients with pre-existing diseases, most frequent hypertension and diabetes, but hypertension, chronic lung disease, and cardiovascular disease presented higher risk. A total of 13% were hospitalized in Intensive Therapy Unit. The mortality of the cohort was 9.77%. Mortality was higher for patients aged 65 or more (OR 5.09), and for those had some pre-existing disease (OR 2.61). Our observations are consistent with reports demonstrating older persons, and those with comorbidities have the highest risk of mortality related to COVID-19. However, unlike other reports from developed or some developing countries, the mortality in our study is lower. This finding may be related to age of our cohort is younger than other published. Also, the health system was able to respond to the demand.

Evaluation of the safety profile of the vaccine candidate Brucella melitensis 16MΔvjbR strain in goats.

Small ruminant brucellosis is caused by the Gram negative cocci-bacillus Brucella (B.) melitensis, the most virulent Brucella species for humans. In goats and sheep, middle to late-term gestation abortion, stillbirths and the delivery of weak infected offspring are the characteristic clinical signs of the disease. Vaccination with the currently available Rev. 1 vaccine is the best option to prevent and control the disease, although it is far from ideal. In this study, we investigate the safety of the B. melitensis 16MΔvjbR strain during a 15-month period beginning at vaccination of young goats, impregnation, delivery and lactation. Forty, 4 to 6 months old, healthy female crossbreed goats were randomly divided into four groups (n = 10) and immunized subcutaneously with a single vaccine dose containing 1x109 CFU of B. melitensis 16MΔvjbR delivered in alginate microcapsules or non-encapsulated. Controls received empty capsules or the commercially available Rev.1 vaccine. Seven months post-vaccination, when animals were sexually mature, all goats were naturally bred using brucellosis-free males, and allowed to carry pregnancies to term. Blood samples to assess the humoral immune response were collected throughout the study. At two months post-delivery, all dams and their offspring were euthanized and a necropsy was performed to collect samples for bacteriology and histology. Interestingly, none of the animals that received the vaccine candidate regardless of the formulation exhibited any clinical signs associated with vaccination nor shed the vaccine strain through saliva, vagina or the milk. Gross and histopathologic changes in all nannies and offspring were unremarkable with no evidence of tissue colonization or vertical transmission to fetuses. Altogether, these data demonstrate that vaccination with the mutant strain 16MΔvjbR is safe for use in the non-pregnant primary host.

Environmental factors modulated ancient mitochondrial DNA variability and the prevalence of rheumatic diseases in the Basque Country.

Among the factors that would explain the distribution of mitochondrial lineages in Europe, climate and diseases may have played an important role. A possible explanation lies in the nature of the mitochondrion, in which the energy generation process produces reactive oxygen species that may influence the development of different diseases. The present study is focused on the medieval necropolis of San Miguel de Ereñozar (13th-16th centuries, Basque Country), whose inhabitants presented a high prevalence of rheumatic diseases and lived during the Little Ice Age (LIA). Our results indicate a close relationship between rheumatic diseases and mitochondrial haplogroup H, and specifically between spondyloarthropathies and sub-haplogroup H2. One possible explanation may be the climate change that took place in the LIA that favoured those haplogroups that were more energy-efficient, such as haplogroup H, to endure lower temperatures and food shortage. However, it had a biological trade-off: the increased risk of developing rheumatic diseases.

Pelvic exenteration for gynecologic malignancies: Postoperative complications and oncologic outcomes. / Exenteración pélvica para neoplasias ginecológicas: Complicaciones postoperatorias y resultados oncológicos.

INTRODUCTION AND OBJECTIVE: To evaluate complications, morbidity and oncologic outcomes of pelvic exenteration as treatment for gynecologic malignancies. MATERIALS AND METHODS: Between 2008 and 2015, a total of 35 patients underwent pelvic exenteration, due to recurrence of gynecological cancer. Surgical outcomes, early and late postoperative complications, and recurrence/survival outcomes were assessed. RESULTS: Mean patient age was 53.8 years. Anterior exenteration was done in 20 patients, while 15 were total exenterations. Ileal conduit was done in 24 patients, while 8 received a neobladder and 3 a cutaneous ureterostomy. Postoperative complications were divided in 2groups, early (<30 days) and late complications (>30 days). A total of 25 patients (71.4%) had one or more early complications; 16 (45.7%) had fever due to a urinary tract infection, pyelonephritis or intra-abdominal collection; 2 (5.7%) developed a vesicovaginal fistula; 4 (11.4%) a rectovaginal fistula; 3 (8.5%) acute kidney failure and one (2.85%) uronephrosis. Regarding to late complications, 8patients (22.8%) had fever. Six (17%) presented with uronephrosis, and 5 (14.2%) with ureteral-pouch stricture. Five patients (14.2%) had acute renal insufficiency, 3 (8,6%) rectovaginal fistula and one (2.85%) urinary fistula. Mean follow up time was 20.3 month (2-60). A total of 22patients (62.8%) were free of disease. Another 13 (37.1%) patients relapsed. Only 4 (11.4%) patients died after pelvic exenteration due to underlying disease. CONCLUSION: Pelvic exenteration has a high rate of complications and morbidity, but can be the last curative opportunity in patients with recurrent or persistent gynecologic malignancies. This procedure should be performed by multidisciplinary, experienced teams in a tertiary medical center.

Efficacy and safety of withholding antimicrobial treatment in children with cancer, fever and neutropenia, with a demonstrated viral respiratory infection: a randomized clinical trial.

OBJECTIVES: To determine efficacy and safety of withholding antimicrobials in children with cancer, fever and neutropenia (FN) with a demonstrated respiratory viral infection. METHODS: Prospective, multicentre, randomized study in children presenting with FN at five hospitals in Santiago, Chile, evaluated at admission for diagnosis of bacterial and viral pathogens including PCR-microarray for 17 respiratory viruses. Children positive for a respiratory virus, negative for a bacterial pathogen and with a favourable evolution after 48 h of antimicrobial therapy were randomized to either maintain or withhold antimicrobials. Primary endpoint was percentage of episodes with uneventful resolution. Secondary endpoints were days of fever/hospitalization, bacterial infection, sepsis, admission to paediatric intensive care unit (PICU) and death. RESULTS: A total of 319 of 951 children with FN episodes recruited between July 2012 and December 2015 had a respiratory virus as a unique identified microorganism, of which 176 were randomized, 92 to maintain antimicrobials and 84 to withdraw. Median duration of antimicrobial use was 7 days (range 7-9 days) versus 3 days (range 3-4 days), with similar frequency of uneventful resolution (89/92 (97%) and 80/84 (95%), respectively, not significant; OR 1.48; 95% CI 0.32-6.83, p 0.61), and similar number of days of fever (2 versus 1), days of hospitalization (6 versus 6) and bacterial infections throughout the episode (2%-1%), with one case of sepsis requiring admission to PICU in the group that maintained antimicrobials, without any deaths. CONCLUSIONS: The reduction of antimicrobials in children with FN and respiratory viral infections, based on clinical and microbiological/molecular diagnostic criteria, should favour the adoption of evidence-based management strategies in this population.

Carboxyl ester hydrolase from Penicillium expansum: cloning, characterization and overproduction by Penicillium griseoroseum.

AIMS: In this study, a gene that encodes a carboxylesterase (carb) in Penicillium expansum GF was cloned, sequenced and overexpressed by Penicillium griseoroseum PG63, and the enzyme was characterized. METHODS AND RESULTS: The recombinant strain, P. griseoroseum T55, obtained upon transformation using the plasmid pAN-52-1-carb, showed integration of the carb gene into at least two heterologous sites of the genome by Southern blotting. Furthermore, the recombinant strain T55 exhibited almost a fourfold increase in carboxylesterase activity compared with PG63 strain when both were cultured without inducers. Based on the secondary structure and multiple sequence alignments with carboxylesterases, cholinesterase and lipase, a three-dimensional model was obtained. The α/ß barrel topology, that is typical of esterases and lipases, was indicated for the CARB protein with Ser(213)-Glu(341)-His(456) as the putative catalytic triad. CARB preferentially hydrolysed acyl chains with eight carbon atoms, and its activity was optimal at a pH of 7·0 and a temperature of 25°C. CARB exhibited stability in alkaline pH, high activity under mesophilic conditions and stability in organic solvents. CONCLUSION: The CARB protein is potentially useful in bioremediation, food and chemical/pharmaceutical industries. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to report the development of a recombinant strain superproducing a Penicillium sp. carboxylesterase.

Prospective evaluation of a model of prediction of invasive bacterial infection risk among children with cancer, fever, and neutropenia.

A risk prediction model for invasive bacterial infection (IBI) was prospectively evaluated among children presenting with cancer, fever, and neutropenia. The model incorporated assessment of 5 previously identified risk factors: serum level of C-reactive protein (CRP) >/=90 mg/L, hypotension, identification of relapse of leukemia as the cancer type, platelet count of

Prospective, multicenter evaluation of risk factors associated with invasive bacterial infection in children with cancer, neutropenia, and fever.

PURPOSE: To identify clinical and laboratory parameters present at the time of a first evaluation that could help predict which children with cancer, fever, and neutropenia were at high risk or low risk for an invasive bacterial infection. PATIENTS AND METHODS: Over a 17-month period, all children with cancer, fever, and neutropenia admitted to five hospitals in Santiago, Chile, were enrolled onto a prospective protocol. Associations between admission parameters and risk for invasive bacterial infection were assessed by univariate and logistic regression analyses. RESULTS: A total of 447 febrile neutropenic episodes occurred in 257 children. Five parameters were statistically independent risk factors for an invasive bacterial infection. Ranked by order of significance, they were as follows: C-reactive protein levels of 90 mg/L or higher (relative risk [RR], 4.2; 95% confidence interval [CI], 3.6 to 4.8); presence of hypotension (RR, 2.7; 95% CI, 2.3 to 3.2); relapse of leukemia as cancer type (RR, 1.8, 95% CI, 1.7 to 2.3); platelet count less than or equal to 50,000/mm(3) (RR, 1.7; 95% CI, 1.4 to 2.2); and recent (< or = 7 days) chemotherapy (RR, 1.3; 95% CI, 1.1 to 1.6). Other previously postulated risk factors (magnitude of fever, monocyte count) were not independent risk factors in this study population. CONCLUSION: In a large population of children, common clinical and laboratory admission parameters were identified that can help predict the risk for an invasive bacterial infection. These results encourage the possibility of a more selective management strategy for these children.

[Causative agents of bloodstream infections in children with neoplasm, in 5 hospitals of Santiago (1994-1998)]. / Agentes causantes de infecciones del torrente circulatorio en niños con cáncer, en cinco hospitales de Santiago (1994-1998).

BACKGROUND: Pediatric patients in treatment for cancer can have fatal bacterial infections. Thus, in the presence of fever or other signs infection, antimicrobials have to be prescribed empirically. AIM: To know the causative agents of bacteremia in children with cancer, their changes with time and between different hospitals and their patterns of susceptibility. MATERIAL AND METHODS: We reviewed the blood cultores of children with cancer in five hospitals of Santiago, from 1994 at 1998. RESULTS: During the study period, 707 agents were isolated. The most frequently isolated species or genus were coagulase negative Staphylococcus (43%), Staphylococcus aureus (16%), Escherichia coli (9%), Klebsiella spp. (8%), Pseudomonas spp. (5%) and Candida spp. (4%). Coagulase negative Staphylococcus was 55% resistant to meticilin and S. aureus was 44% resistant. Enterobacteriaceae had 15% resistance to gentamicin and amikacin, 2% to imipenem, 26% to ceftriaxone, 21% to cefotaxime and 20% to ceftazidime. Among non fermenting agents resistance was 6% for imipenem, 9% for amikacin 10% for ciprofloxacin, 19% for ceftazidime and 22% for cefoperazone. The resistance of Streptococcus spp. (non pneumoniae) to penicillin reached 50% and that of Enterococcus spp. was of 33%. CONCLUSIONS: Treatment for pediatric patients with cancer must be modified and new guidelines including more active medications for patients at risk for bacteremia, should be devised.

Diurnal variations of opioid peptides and synenkephalin in vitro release in the amygdala of kindled rats.

Pentylenetetrazol (PTZ) kindling was induced in male Wistar rats (250-300 g) by daily intraperitoneal injections of 35 mg/kg of the convulsant agent. Immunoreactive (IR)-Met-enkephalin (IR-ME), IR-Leu-enkephalin (IR-LE), IR-heptapeptide (IR-HE), IR-octapeptide (IR-OC) and IR-synenkephalin (IR-Syn) in vitro release was measured from amygdala slices 24 h after the last stimulus, in groups of eight rats, every 4 h beginning at 08:00 h. Opioid peptides in vitro release displayed diurnal variations. IR-ME and IR-Syn showed maximal levels before the onset of darkness (16:00 h). IR-LE and IR-OC release was enhanced 4 h later (20:00 h), no changes were detected for IR-HE. These results show that endogenous opioid system (EOS) release displays diurnal variations. The peak for the analysed peptides was reached before and during the dark phase. It is suggested that EOS release enhancement in PTZ-kindled rats, seems to be due to a compensatory mechanism against the excitation induced by the blockade of the GABAergic transmission.

Valproic acid-induced rapid changes of met-enkephalin levels in rat brain. Probable association with abstinence behavior and anticonvulsant activity.

Valproic acid (VPA) induces abstinence behavior and analgesia and displays an anticonvulsant effect, but its exact mechanism of action is not yet clear. In order to view whether proenkephalin derived-peptides are involved in the mechanism of VPA-induced behavior, we analyzed immunoreactive-met-enkephalin (IR-ME) in rat striatum, midbrain, and amygdala 10, 20, and 45 min after i.p. injection of 200 mg/kg of VPA. VPA induced body shakes that peaked within 5 to 10 min. IR-ME increased in the striatum and decreased in the midbrain at 10, 20, and 45 min, reaching the highest and lowest levels at 10 and 20 min, respectively. No changes occurred in the amygdala. Gel filtration chromatography followed by HPLC of striatum extracts showed that the increased IR-ME levels corresponded to low molecular weight peptides, including ME. These results indicate that VPA produced rapid changes of IR-ME levels in rat brain and suggest peptide participation in the mechanisms of VPA-induced behavior. The anticonvulsant effect of VPA was tested in rats treated with pentylenetetrazol (70 mg/kg) 30 min after VPA (400 mg/kg) administration, and IR-ME was analyzed in striatum 15 min later. No changes in striatal IR-ME levels occurred in protected rats (no behavioral convulsions), compared with those treated only with VPA, but a significant decrease appeared in unprotected animals (clonic convulsions). These results suggest that striatal ME may participate in the mechanism of VPA-induced abstinence behavior and in the anticonvulsant effect. Otherwise, midbrain ME might be involved in other VPA behaviors such as analgesia.

IR-Met and IR-Leu enkephalin content in the axolotl brain (Ambystoma mexicanum).

The peptides met- and leu-enkephalin were identified in the telencephalon, rombencephalon, diencephalon and hypophysis of Ambystoma mexicanum brain by radioimmunoassay procedure. The met-enkephalin was the predominant peptide present in the axolotl brain in contrast with leu-enkephalin, except in the hypophysis where the ratio MET/LEU was 2.2/l. The clear differences in the concentration between enkephalins through a submammalian brain species as Ambystoma genus and the possibility that leu-enkephalin is derived exclusively from a precursor like prodynorphin offers an excellent model for the opioids biosynthetic processes.

Diurnal variations of IR-Met-enkephalin in the brain of pentylenetetrazol-kindled rats.

Pentylenetetrazol (PTZ) kindling was induced in male Wistar rats by daily i.p. injections of 40 mg/kg of the convulsant agent. Immunoreactive (IR)-Met-enkephalin was quantified in the amygdala, hippocampus and hypothalamus 17 days after the last stimulus, in groups of 6-7 rats, every 4 h, beginning at 08.00 h. IR-Met-enkephalin level displayed diurnal variations in brain regions of control animals. In the amygdala and the hippocampus the peptide peaked at 24.00 h and in the hypothalamus at 20.00 h; the troughs were at 08.00, 16.00 and 08.00 h, respectively. Diurnal variations were abolished in the amygdala and hypothalamus of kindled rats. In the amygdala the effect was characterized by an IR-Met-enkephalin increase at 04.00, 08.00 and 12.00 h; in the hypothalamus the peptide was enhanced at 08.00 and 12.00 h; in the hippocampus IR-Met-enkephalin increased at 12.00 h and showed a displacement of the peak during the dark phase. The results suggest that PTZ kindling in rats produces a long-lasting alteration on diurnal variations of IR-Met-enkephalin levels in limbic structures.

Pentylenetetrazol-induced seizures produce an increased release of IR-Met-enkephalin from rat striatum in vitro.

In this work we analyzed the immunoreactive-methionine-enkephalin (IR-Met-enkephalin) levels in several brain regions of rats sacrificed during the tonic extension, induced by acute treatment with pentylenetetrazol (PTZ). The results show an increased of IR-Met-enkephalin content in striatum but not in amygdala, hypothalamus, septum, hippocampus and cortex. To characterize whether this elevation of enkephalin levels in striatum corresponded to the releasable pool, we studied the in vitro efflux of this peptide in striatal slices of rats sacrificed during the seizures, in acute PTZ and in PTZ-kindled rats (kindling group I). In addition, PTZ-kindled rats were analyzed 24 h after the last stimulus (kindling group II). The striatal slices of acute group and kindling group I displayed a significant increase in the evoked release of IR-Met-enkephalin. However, no significant changes occurred from striatal slices of kindling group II animals. In vitro superfusion of GABA (100 microM) produced a approximately equal to 63% decrease in IR-Met-enkephalin released from striatal slices in both saline and acute PTZ-treated rats. Several studies suggest that opioid peptides may be released in the ictal phase of seizure in order to mediate some transient postictal behavior. Our results suggest that of several brain regions tested, only the striatal IR-Met-enkephalin may be released during the ictus to mediate postictal behavior in the acute PTZ treated and in PTZ-kindled rats. This effect may be regulated by the GABA system.

Pentylenetetrazol kindling produces a long-lasting elevation of IR-Met-enkephalin but not IR-Leu-enkephalin in rat brain.

Pentylenetetrazol (PTZ) kindling was induced in male Wistar rats by daily i.p. injections of 40 mg/kg of the convulsant agent. IR-Met-enkephalin and IR-Leu-enkephalin were quantified in several brain regions 16 days after the last stimulus. In addition other rats received another PTZ dose on the sixteenth day and the samples were analyzed 1 and 24 h later. The results showed a long-lasting elevation in amygdala, septum, hypothalamus and hippocampus of IR-Met-enkephalin only. These brain structures also showed a decrease of IR-Met-enkephalin 1 h after the PTZ-induced seizure, but reached newly elevated levels 24 h later. IR-Leu-enkephalin levels only showed a decrease in striatum 1 h after PTZ-induced convulsions. The data suggest that Met-enkephalin is related to permanent changes in brain function induced by PTZ kindling, while Leu-enkephalin may depend on the occurrence of epileptic seizures.

Brain content of immunoreactive [Leu5]enkephalin and [Met5]enkephalin after pentylenetetrazol-induced convulsions.

Wistar rats were injected daily with convulsant doses of PTZ (40 mg/kg). The brain content of immunoreactive (IR) enkephalins was measured after repetitive clonic convulsions and after the 'kindling' state was reached. In both instances we found a significant increase of IR [Leu5]enkephalin and IR [Met5]enkephalin in septum, striatum and amygdala 24 h after PTZ administration. These results support the suggestion that the peptide increase occurs with repetitive convulsions and not only after the 'kindling' state is reached.