Expression and novel alternative purification of the recombinant nucleocapsid (N) protein of SARS-CoV-2 in Escherichia coli for the serodiagnosis of COVID-19

The SARS-CoV-2 coronavirus causes severe acute respiratory syndrome and has caused a global pandemic by causing the COVID-19 disease. To monitor and control it, diagnostic methods such as molecular and serological tests are necessary. The serological approach uses SARS-CoV-2 antigens to detect the antibodies present in patients using quantitative techniques such as enzyme-linked immunosorbent assay (ELISA) or qualitative rapid tests such as lateral flow chromatography (RDTs). The main antigens used are the spike protein (S) and the nucleocapsid protein (N). Both proteins are obtained in different expression systems, in eukaryotic cells, their production is expensive, so in this work we chose a simpler and cheaper system such as prokaryotic to express and purify the N protein. Thereore, the nucleotide sequence had to being optimized to be expressed in Escherichia coli. The protein N is sensitive to E.coli proteases and also has the ability to self-proteolyze under native conditions, degrading into different fragments. However, under denaturing conditions, using urea and at pH 5.3 it is stable and efficiently purified using metal exchange chromatography (IMAC). In our purification strategy, we surprisingly found that by not using a sonicator, a homogeneous and time-stable preparation of the recombinant antigen is obtained. An approximate yield of 200 mg / L was obtained. It was then tested with healthy sera and sera from COVID-19 convalescent patients in Wester-blot tests that were able to recognize it. Our work provides a novel strategy to produce the SARS-CoV-2 protein N so that it can be used as an input in the development and innovation of serological tests in the diagnosis of COVID-19.

Housing Conditions Linked to Tick (Ixodida: Ixodidae) Infestation in Rural Areas of Colombia: A Potential Risk for Rickettsial Transmission.

This cross-sectional study explores the different conditions related to the infestation of ticks in households and the potential risks for rickettsial transmission in Urabá, Colombia. The main outcome of interest was villagers' perception of tick infestation. The data were analyzed using a clog-log mixed regression model. Ticks were collected from infested humans to diagnose infection by spotted fever group rickettsiae (SFGR). In addition, a thematic analysis of qualitative data from key informants concerning knowledge about ticks was conducted. The prevalence of infestation of ticks in households was estimated at 60.99% (95% CI: 51.58-93.51). The multivariate model suggested that households with palm leaf roofs (PR = 1.95; 95% CI: 1.19-2.95), canines (PR = 1.76; 95% CI: 1.21-2.46), rats (PR = 2.19; 95% CI: 1.45-3.08), and with the presence of opossums in areas surrounding the households (PR = 1.51; 95% CI: 1.05-2.10) had a higher prevalence of tick infestation. Two samples of the tick species Amblyomma patinoi were found infected with Rickettsia amblyommatis and Candidatus Rickettsia colombianensi. A thematic analysis provided the names that local community members give to ticks, areas where ticks are common, and the individuals at risk of infestation. The presence of domestic, synanthropic, and wild animals suggests a high risk of the dissemination of ticks inside dwellings and close to them in these rural areas.

Eco-epidemiological analysis of rickettsial seropositivity in rural areas of Colombia: A multilevel approach.

Rickettsiosis is a re-emergent infectious disease without epidemiological surveillance in Colombia. This disease is generally undiagnosed and several deadly outbreaks have been reported in the country in the last decade. The aim of this study is to analyze the eco-epidemiological aspects of rickettsial seropositivity in rural areas of Colombia where outbreaks of the disease were previously reported. A cross-sectional study, which included 597 people living in 246 households from nine hamlets in two municipalities of Colombia, was conducted from November 2015 to January 2016. The survey was conducted to collect sociodemographic and household characteristics (exposure) data. Blood samples were collected to determine the rickettsial seropositivity in humans, horses and dogs (IFA, cut-off = 1/128). In addition, infections by rickettsiae were detected in ticks from humans and animals by real-time PCR targeting gltA and ompA genes. Data was analyzed by weighted multilevel clog-log regression model using three levels (person, household and hamlets) and rickettsial seropositivity in humans was the main outcome. Overall prevalence of rickettsial seropositivity in humans was 25.62% (95%CI 22.11-29.12). Age in years (PR = 1.01 95%CI 1.01-1.02) and male sex (PR = 1.65 95%CI 1.43-1.90) were risk markers for rickettsial seropositivity. Working outdoors (PR = 1.20 95%CI 1.02-1.41), deforestation and forest fragmentation for agriculture use (PR = 1.75 95%CI 1.51-2.02), opossum in peridomiciliary area (PR = 1.56 95%CI 1.37-1.79) and a high proportion of seropositive domestic animals in the home (PR20-40% vs <20% = 2.28 95%CI 1.59-3.23 and PR>40% vs <20% = 3.14 95%CI 2.43-4.04) were associated with rickettsial seropositivity in humans. This study showed the presence of Rickettsia antibodies in human populations and domestic animals. In addition, different species of rickettsiae were detected in ticks collected from humans and animals. Our results highlighted the role of domestic animals as sentinels of rickettsial infection to identify areas at risk of transmission, and the importance of preventive measures aimed at curtailing deforestation and the fragmentation of forests as a way of reducing the risk of transmission of emergent and re-emergent pathogens.

Dexamethasone does not reduce length of hospitalization or recurrent wheezing 1 year after early bronchiolitis.

AIM: It has been suggested that a 6-day dexamethasone course combined with adrenaline may significantly reduce the admission rate in outpatients. It has also been suggested that prednisolone may reduce recurrent wheezing. Our aim was to investigate whether treatment with steroids, especially high dose dexamethasone, is associated with the length of hospitalization or with recurrent wheezing. METHODS: A prospective-retrospective observational study was performed from January 2009 to December 2011 in a secondary care hospital. Eighty previously healthy patients aged 7-180 days who were hospitalized with a first episode of acute bronchiolitis (AB) were studied. AB treatment was at the attending physician's discretion, among those tested for AB with some benefit. Eligible drugs included bronchodilators and steroids. Primary short-term outcome studied was days of hospitalization. Primary long term outcome was "recurrent wheezing". RESULTS: Median length of hospitalization was 6 days for all groups. Patients treated with dexamethasone (1 mg/kg/day for 1 day, plus 0.6 mg/kg/day for 5 days) had an average length of hospitalization of 6.8 ± 3.1 (range, 4-20) days, those on prednisolone (prednisolone 1-2 mg/kg/day for 5 days) 7.0 ± 2.6 (range, 4-12) days and those with no steroids 6.6 ± 3.3 (3-21) days (no significant differences). The analysis showed no evidence of association of any management schedule with recurrent wheezing. After adjusting long-term outcome variables for potential confounders, comparisons remained no different. CONCLUSION: High dose dexamethasone or medium-dose prednisolone during AB provided no benefit in a short or long-term period in young, previously healthy hospitalized infants.

Trombo de Gran Tamaño en Auricula Derecha Simulando un Mixoma / Large Thrombus at Right Artium Mimicking a Myxoma

La presencia de trombos en aurícula derecha es infrecuente y muy poco frecuente en corazones estructuralmente normales. Presentamos el caso de un paciente cuyo diagnóstico ecocardiografico definitivo, un trombo de gran tamaño en aurícula derecha, ofrecía dificultades debido a las características ecocardiograficas de la masa intracardiaca, que no obstante sus dimensiones provocaba escasa sintomatología y simulaba un mixoma auricular(AU)

Descripción epidemiológica de la infección con agentes del género rickettsia en roedores, ectoparásitos y humanos en el Urabá antioqueño

Rickettsia es un género de bacterias trasmitidas por ectoparásitos hematófagos.El primer reporte de Rickettsiosis en Colombia data de la década de los años 30, cuando se describió una enfermedad febril con brote eruptivo en piel, en la población de Tobia Cundinamarca.Los últimos brotes fueron diagnosticados en los municipios de Necoclí (Antioquia), Los Córdobas(Córdoba) y Turbo (Antioquia), en los años 2006, 2007 y 2008 respectivamente (1-3). El presente estudio pretendió realizar una descripción epidemiológica de la infección con bacterias del género Rickettsia en roedores, ectoparásitos y humanos en el Urabá antioqueño.

Long-term results of the Spanish Protocol SO-95 for the treatment of non-metastatic high-grade osteosarcoma of the extremities in children

INTRODUCTION: The long-term results of the Spanish Study Protocol SEOP-SO-95 for treatment of localised osteosarcoma of the extremities in children were evaluated. PATIENTS AND METHODS: One hundred consecutive patients under 18 years of age from 22 institutions were enrolled from January 1995 to December 2000. Immunohistochemical expression of p53, HER/erbB-2 and P-glycoprotein were retrospectively studied in 27 patients. Treatment consisted of: preoperative chemotherapy with doxorubicin, cisplatin, high-dose methotrexate with leucovorin rescue and ifosfamide for 14 weeks; surgery of primary tumour in week 16; postoperative chemotherapy with the above-mentioned drugs for 25 weeks. RESULTS: With a median follow-up of 124 months (range 84-158 months), 69 patients (69%) were continuously event-free survivors; the 10-year probability of event-free survival (EFS) was 62%. Conservative surgery was performed in 85% of patients. Twenty-six patients had local recurrence or distant relapse. The median time to recurrence/ relapse was 27 months (range 17-93 months). The local recurrence rate was 7% (7 of the 100 patients); 4 had wide surgical margins, 2 marginal and 1 intralesional. Four patients died as a result of chemotherapy-related toxicity and 1 developed a second neoplasia (acute myeloid leukaemia). p53 expression and HER2/erbB-2 expression showed no effect on survival or EFS. CONCLUSIONS: This therapeutic protocol achieved good oncologic and orthopaedic results. We observed a significant treatment-related toxicity (AU)

Cervical length and the risk of spontaneous labor at term.

OBJECTIVE: To investigate the relationship between cervical length (CL) at 37 to 40 weeks and delivery within 7 days and delivery by 41 weeks. STUDY DESIGN: We performed transvaginal ultrasound to measure CL in women with singleton gestations at 37 to 40 weeks. We then used a receiver operating characteristic curve (ROC) to assess the relationship between CL and delivery within 7 days and delivery by 41 weeks. RESULT: For the 120 women included in the analysis, the mean CL (+/-s.d.) was 25.3+/-9.8 mm. The logistic regression model to predict each of the outcomes includes gestational age at ultrasound (GA-US) and CL. Neither birthweight, nor parity seems to affect the probability of delivery within 7 days. The ROC curve was used to assess the probability of spontaneous labor within 7 days at each CL measurement. The likelihood ratio of delivery within 7 days when CL is < or = 10 mm is 12. CONCLUSION: CL measurement at 37-40 weeks is an independent predictor of delivery within 7 days and delivery by 41 weeks regardless of GA-US. This information can be utilized when counseling patients regarding the management of term pregnancies.

Antenatal exposure to magnesium sulfate and the incidence of patent ductus arteriosus in extremely low birth weight infants.

OBJECTIVE: Magnesium sulfate (MgSO(4)) is the most commonly used tocolytic agent in the US and is also employed as a prophylactic agent against seizures in pre-eclamptic women. MgSO(4) crosses the placenta and its concentration in the newborn usually exceeds that of maternal levels. The purpose of this study was to explore the relationship between antenatal exposure to MgSO(4) and the incidence of patent ductus arteriosus (PDA) in extremely low birth weight infants. STUDY DESIGN: A total of 954 neonates with birth weights between 500 and 1000 g, born at the University of Miami/Jackson Memorial Hospital between January 1995 and December 2004 and surviving for more than 3 days, were followed until death or discharge from the hospital. The incidence of PDA in infants exposed to MgSO(4) was compared with those not exposed and comparisons were also made between infants exposed to different maternal doses of MgSO(4). RESULTS: The incidence of PDA was significantly higher in the group of infants exposed to MgSO(4) compared with the unexposed control group (67 vs. 60%, P<0.018). When stratified by gestational age the differences were significant only in the group of infants with a gestational age of >or=26 weeks (58 vs. 49%, P<0.039). Logistic regression analysis to adjust for co-variables indicated an increased risk of PDA with higher doses of MgSO(4) (odds ratio 1.33 confidence interval (CI) 1.12 to 1.58, per 50 g of MgSO(4)). CONCLUSION: Antenatal exposure to MgSO(4) is associated with a higher risk of PDA in extremely low birth weight infants and this effect is more significant and dose-related in more mature infants.

Birth weight references for triplets.

OBJECTIVE: The purpose of this study was to formulate growth references that reflect triplet fetal and neonatal populations at each gestational age by combining serial ultrasonographic estimates of fetal weights and measured birth weights. STUDY DESIGN: This historical cohort study was based on 188 pregnancies of live-born triplets of > or =23 weeks' gestation. Ultrasonographic fetal weight measures were modeled as a function of gestational age for each infant. Linear regression models were used to fit the data, and weight percentiles were generated. RESULTS: Well-grown triplets fell substantially below singletons by 30 weeks and twins after 34 weeks. Trichorionic vs monochorionic or dichorionic placentation resulted in 27% higher growth at the 10th %ile, 5% higher growth at the 50th %ile, and 4% higher growth at the 90th %ile by 34 weeks. CONCLUSION: The overall pattern of fetal growth for well-grown triplets does not differ from that of singletons and twins until late gestation, confirming that, in utero, well-grown children have similar growth potentials, regardless of plurality.

The Zavanelli maneuver in two cases of shoulder dystocia.

Several reports describe the replacement of the partially delivered fetus into the vagina followed by Cesarean section after failed vaginal delivery. Undeliverable shoulder dystocia is by far the most common cause of failed vaginal delivery. The objective of this report was to review the safety and to reconsider when to perform the Zavanelli maneuver in the management of shoulder dystocia. We report two cases of severe shoulder dystocia in which commonly recommended therapeutic options failed. Cephalic replacement was performed with ease followed by Cesarean section. Endometritis was the maternal complication in both cases. Both infants had transient Erb's palsy. Although traditionally the Zavanelli maneuver is a method of last resort in the management of shoulder dystocia, consideration should be given to its use earlier in the treatment of obstructed vaginal delivery.

In utero demonstration of aberrant systemic blood supply in lung sequestration: a case report

Prenatal diagnosis of a potentially lethal condition as shown and confirmed with color flow Doppler; demonstration of an aberrant systemic blood supply

Allogeneic peripheral blood stem cell transplantation (PBSCT) from HLA-identical sibling donors in children with hematological diseases: a single center pilot study.

Between February 1995 and July 1999 25 pediatric patients (8 months to 14 years old) underwent peripheral blood stem cell transplantation (PBSCT) from an HLA-identical sibling donor. Diagnoses included ALL (17), non-ALL (6), and non-malignant disease (2). GVHD prophylaxis consisted of cyclosporine plus methotrexate (15), only cyclosporine (8), cyclosporine plus prednisone (1), or nothing (1). All donors (6 months to 41 years old) received G-CSF at 10 microg/kg/day subcutaneously for 4-5 days and on day 5 underwent large volume leukapheresis. Median number of CD34(+) and CD3(+) cells collected and infused was 6.9 x 10(6) (range 2.5-32.8) and 4.5 x 10(8) (0.5-22.1) per kg of recipient body weight respectively. Median time to achieve ANC >0.5 x 10(9)/l and platelets >20 x 10(9)/l was 10 and 12 days, respectively. Acute GVHD grade > or =II developed in 10 of 24 evaluable patients (42%). Probability of acute GVHD was 62%. Median time to discharge was 25 days (range 14-52). Among 20 evaluable patients, five (25%) developed chronic GVHD at day 100. Probability of chronic GVHD was 29% after 1 year post PBSC. At a median follow-up of 558 (9-2071) days, overall survival for the whole group is 68%. Probabilities of event-free survival, overall survival and relapse for patients with malignant hematological diseases are 53%, 59% and 24% at 5 years, respectively. This study has confirmed the feasibility and safety of mobilization and collection of PBSC products and the applicability of this procedure to the pediatric population, both donors and recipients. Studies including larger numbers of pediatric patients undergoing allogeneic PBSCT are warranted to determine the long-term outcomes of such procedures.

Ki-1+ anaplastic large cell lymphoma in a child with unpredictable clinical course.

Ki-1+ anaplastic large cell lymphoma (Ki-1+ ALCL) is a subtype of non-Hodgkin lymphoma (NHL) with defined histopathological characteristics but with highly variable clinical presentation and outcome. Although in most of the patients the disease behaves as an intermediate- or high-grade lymphoma, some patients present with an indolent clinical course. Factors that determine the clinical behavior of this lymphoma have not yet been identified. A case is reported of a 13-year-old girl who initially presented with Ki-1+ ALCL but later developed recurrent localized cutaneous disease and followed a clinical course similar to that of a low-grade lymphoma.

In utero demonstration of aberrant systemic blood supply in lung sequestration: a case report.

Prenatal diagnosis of a potentially lethal condition as shown and confirmed with color flow Doppler; demonstration of an aberrant systemic blood supply.

Clinical and economic comparison of allogeneic peripheral blood progenitor cell and bone marrow transplantation for acute lymphoblastic leukemia in children.

There is limited experience in the use of peripheral blood progenitor cells (PBPC) for allogeneic transplantation in children. In the present study we compared engraftment kinetics, incidence of acute and chronic graft-versus-host disease (GVHD) and the outcome and economic costs of allogeneic PBPCT vs BMT in children with ALL in a single institution. All children were transplanted in complete remission (CR) with a similar conditioning regimen and the same GVHD prophylaxis. Patients undergoing PBPCT achieved myeloid and platelet engraftment before patients undergoing BMT (P < 0.001). Platelet recovery was faster for the PBPCT group (P < 0.014 for 50 x 10(9)/l and P < 0.039 for 100 x 10(9)/l). Incidence and severity of acute and chronic GVHD were similar in both groups (acute grade 1-2: 9/13 for PBPCT vs 9/11 for BMT; chronic GVHD: 5/12 for PBPCT vs 3/8 for BMT). Hospital stay was shorter for the PBPCT than for the BMT group (28.8 days vs 42.9 days, respectively) and the PBPCT group used less clinical resources, resulting in overall lower cost for PBPCT (US $14,046) compared to BMT (US $19,840). There was no statistically significant difference in DFS between PBPCT and BMT (68.4% vs 50%, respectively).

Use of concurrent G-CSF + GM-CSF vs G-CSF alone for mobilization of peripheral blood stem cells in children with malignant disease.

There is limited experience in the mobilization of peripheral blood progenitor cells (PBPC) in children and the optimal method for PBPC mobilization is unknown. The present study was conducted to ascertain whether mobilization with G-CSF + GM-CSF (group I) provides some advantage over G-CSF alone (group II) in terms of collected CD34+ cells and hematopoietic recovery following myeloablative conditioning in children with malignancies. An economic analysis was also performed. Each group comprised 21 consecutive patients. The mean number of aphereses was 1.5+/-0.5 in group I and 1.2+/-0.46 in group II (NS). The mean number of CD34+ cells was 3.8 x 106+/-4.03/kg in group I and 4.2+/-5.4 in group II (NS). The mean number of total blood volumes (TBV) processed was 4.4+/-1.5 in group I and 4.3+/-1.5 in group II (NS). The mean duration of the procedure was 276+/-74.1 min in group I and 286.7+/-75.9 min in group II (NS), and the inlet flow was 45.1+/-12 ml/min in group I and 39.5+/-15.1 ml/min in group II (NS). No significant differences in the neutrophil and platelet engraftment probability were observed between the two groups. The mean overall cost of group II was not statistically significant from that of group I (US$ 9521+/-330 vs US$ 10201+/-1028, P = NS). The cost of mobilization was significantly higher in group I than in group II, conditioning regimen costs were similar in both groups and the costs related to the post-transplant period were similar in both groups. We conclude that PBPC mobilization with G-CSF + GM-CSF in children does not enhance hematological recovery in comparison with mobilization using G-CSF alone. However, the combination of G-CSF + GM-CSF does not significantly increase the overall cost of transplantation.

[Cerebral malaria in children]. / Neuropaludismo infantil.

INTRODUCTION: Malaria is one of the main health problems in the Third World. Plasmodium falciparum infects as many as 300 million people, causing up to three million deaths each year, most of which occur in African children. Cerebral malaria is the most common lethal complication of P. falciparum infection in children and is defined by three criteria: disturbances of consciousness, presence of P. falciparum parasitaemia and absence of other causes of acute encephalopathy. Cerebral malaria is a medical emergency and parenteral quinine is the most recommended treatment because of the frequency of chloroquine-resistant strains. Mortality is as high as 50 per cent and residual disability is present in about 20 per cent of survivors. OBJECTIVE: We want to warm Spaniard neuropaediatricians about the existence of cases of cerebral malaria in our country in order to get a better diagnose and treatment for those children. PATIENTS AND METHODS: A retrospective medical scores review of 20 hospitalised children diagnosed of malaria from 1990 to 1998. We selected three cases with neurological signs and we analysed clinical onset, EEG, neuroimaging, and permanent sequels. RESULTS: All patients had acute encephalopathy with fever, obtundation and seizures. They all presented residual disability (mainly hemiparesis). CONCLUSION: We must know better about cerebral malaria because of an increasing incidence of imported malaria due to emigration from African countries and Spaniard tourism to areas of endemic paludism.

Patología molecular de las leucemias agudas / Molecular bases of growth pathology

No disponible

Neuropaludismo infantil / Cerebral malaria in children

Introducción. El paludismo es uno de los mayores problemas de salud en los países del Tercer Mundo. El Plasmodium falciparum infecta cada año a unos 300 millones de personas, causando más de tres millones de muertes, principalmente entre los niños africanos. El neuropaludismo es la complicación letal más frecuente de la infección po P. falciparum en el niño. El diagnóstico de malaria cerebral requiere la presencia de tres criterios: alteración del nivel de conciencia, evidencia de parasitemia po P. falciparum y ausencia de otras causas de encefalopatía aguda. El neuropaludismo es una urgencia médica y se recomienda tratar a estos pacientes con quinina por vía parenteral, debido a la alta tasa de plasmodios resistentes a la cloroquina. La mortalidad puede alcanzar cifras de hasta un 50 por ciento y la incidencia de secuelas neurológicas en los supervivientes se sitúa en torno a un 20 por ciento. Objetivo: Llamar la atención de los neuropediatras españoles sobre la presencia de casos de malaria cerebral en nuestro país, lo que redundará en un diagnóstico y un tratamiento más adecuados. Pacientes y métodos. Revisión retrospectiva de las historias clínicas de 20 pacientes diagnósticados de malaria cerebral en nuestro hospital entre 1990 y 1998. Se seleccionaron tres casos con manifestaciones neurológicas. En éstos, se analizan: debut clínico, EEG, hallazgos de neuroimagen y secuelas neurológicas. Resultados. Todos los pacientes presentaron una encefalopatía aguda con fiebre, obnubilación y crisis convulsivas. En todos los casos, existieron secuelas neurológicas permanentes (principalmente, hemiparesia). Conclusión. Es necesario un mejor conocimiento sobre la malaria cerebral ya que existe un aumento de los casos de malaria importada en nuestro país, por una mayor emigración desde los países africanos y por el incremento del turismo español hacia las áreas endémicas (AU)