How molecular diagnosis may modify immunotherapy prescription in multi-sensitized pollen-allergic children.

INTRODUCTION: Specific immunotherapy (SIT) is used to treat asthma and allergic rhinitis, and a dose-response relationship has been found for SIT efficacy, creating a need to accurately select the allergen used in therapy. This need is especially pronounced in poly-sensitized children living in areas where different pollen allergen sources coexist in the same season, as this circumstance complicates diagnostic efforts. In such cases, component-resolved diagnosis (CRD) can increase diagnostic accuracy and aid in SIT prescription. MATERIALS AND METHODS: We hypothesized that CRD results would lead to modifications in classical immunotherapy prescription based on sources such as medical history, season of symptom presentation, and skin testing. We studied a sample of children indicated for immunotherapy in whom classical methods had not pointed out the most relevant allergen due to sensitization to more than two pollens. We used a small panel of recombinant allergens, analyzing the percentage of changes to prescription considering the findings of molecular studies. RESULTS: Of the 70 children included, CRD led to modified immunotherapy prescription in 54.3%. Indications of single-allergen therapy increased from 18% to 51% when CRD was included. The decision to prescribe immunotherapy was reversed following CRD in 9.3% of cases. DISCUSSION: CRD use alters the choice of specific immunotherapy in poly-sensitized children. A wide panel of recombinant allergens may not be necessary to improve immunotherapy indication using molecular techniques; rather, a smaller panel adapted to include those allergens prevalent in the geographical area in question appears to be sufficient for more effective immunotherapy, also leading to an improved cost-benefit ratio.

Obstructive sleep apnea and periodic limb movement in children: is there really an association?

PURPOSE: Periodic limb movements (PLMs) can be found isolated or related to other sleep disorders, as Obstructive Sleep Apnea (OSA). Nevertheless, this association was described before the proposal for modification of the World Association of Sleep Medicine (WASM), which incorporated major changes modifying the definition of respiratory-related leg movements (RRLM) so that the relationship between OSA and PLM could be affected. METHODS: A total of 131 PSG were studied (children with ages from 5 to 12 years old), all referred because of a suspicion of sleep-disordered breathing (65 children were diagnosed of OSA, and 66 presented snoring but no sleep apnea). Leg movements were manually scored according to both 2006 and 2016 WASM/IRLSSG criteria. RESULTS: According to 2006 WASM rules, statistical differences were found, not only for PLM index (p 0.002), but all indexes. Nevertheless, according to new 2016 WASM rules, no statistical differences were found for PLM index (p 0.677), non-REM PLM index (p 0.299), REM PLM index (P 0.511) or PLM with arousal index (p 0.180), between OSA and non-OSA group. Positive correlation between PLM and RRLM have been found with both set of rules. The percentage of children with PLM>5/h is higher when using the prior PLM scoring criteria developed in 2006 (38.93%) versus the updated PLM scoring criteria (19.08%). CONCLUSION: The lack of association when using the new WASM/IRLSSG scoring rules together with the absence of a previous clear etiopathology explanation may suggest that the association between OSA and PLM might be indeed overestimated and that, perhaps, it really did not exist.

Hidden diversity of cestodes in wild African carnivores: I. Non-taeniid cyclophyllideans.

Our knowledge of parasites in wildlife remains limited, primarily due to restricted access to samples, especially of parasites from protected species. This present study contributes to the comprehension of the enigmatic world of helminths of African wild mammals and cestode biodiversity by combining both molecular and morphological analysis. Cestode samples were opportunistically collected from 77 individual definitive hosts in South Africa, Namibia and Ethiopia, encompassing 15 different species of wild African carnivores and additionally domestic cats. The analysis revealed 32 different cyclophyllidean species of which 21 (65.6 %) represent previously unknown genetic entities. They belong to the families Mesocestoididae, Hymenolepididae, Dipylidiidae and Taeniidae. Here we cover the non-taeniid cestodes, while the taeniids will be addressed in a separate publication. Three of the non-taeniid species uncovered in this study could be assigned to the genus Mesocestoides and were isolated from servals and domestic cats. The white-tailed mongoose was found to be a suitable host for a species belonging to the Hymenolepididae, which was identified as Pseudandrya cf. mkuzii. Both feline and canine genotypes of Dipylidium caninum were detected in domestic cats, the canine genotype also in an African wolf. In addition to these, a novel species of Dipylidium was discovered in an aardwolf. Lastly, four distinct species of Joyeuxiella were found in this study, revealing a cryptic species complex and emphasizing the need for a taxonomic reassessment of this genus. Despite the limited scope of our study in terms of geography and sample size, the results highlight that biodiversity of cestodes in African wild mammals is grossly under-researched and follow-up studies are urgently required, in particular linking morphology to gene sequences.

Manual scoring of periodic limb movements in children: is it still necessary?

OBJECTIVE: Although manual scoring has been classically considered the gold standard to identify periodic leg movements (PLM), it is a very time consuming and expensive process, also subject to variability in interpretation. In the last decades, different authors have observed reasonably good agreement between automated PSG scoring algorithms and manual scoring in adults, according to established criteria. We aim to compare the automatic software analysis of our polysomnogram with the manual staging in children with sleep-disordered breathing. METHODS: We performed a semiautomatic method, in which an experienced technician watched the video recording and removed from the automatic analysis those movements that did not correspond to true candidate leg movement (LM). RESULTS: A total of 131 PSGs were studied; applying the established criteria, 65 children were diagnosed of obstructive sleep apnea, and 66 presented snoring but with no sleep apnea. The mean age was 6.7 years (±1.7) and twenty-five children (19.08 %) had a PLMI >5/h. Statistical differences were found not only for PLMI (manual: 2.20 (0.7, 4.1) vs automatic (6.4 (3.85,9.5); p < 0.001), but for almost of all indexes assessed between the automatic and the manual scoring analysis. The level of concordance was only moderate for PLM index (0.63 [0.51-0.72]); showing that, unlike the articles published in the adult population, automatic analysis is not accurate in children and, manually or semi-automatically analysis as ours need to be done. CONCLUSION: It seems that PLM detection algorithm might work accurately but, the real need would be a true LM detection algorithm.

Absence of clinical symptoms upon introduction of egg into the diet of milk-allergic infants not previously sensitised to egg.

BACKGROUND: It is unclear how many children suffering from IgE mediated cow's milk allergy are sensitised to egg in early life and what the clinical implication of this sensitisation is. It is also unclear if those not sensitised to egg in early life, do later on develop sensitisation and clinical allergy to egg. METHODS: This study examines the prevalence of egg sensitisation among infants with allergy to cow's milk, prior to and following the introduction of egg and what this sensitisation clinically means. RESULTS: The percentage of egg-sensitised children seen among the group of children with cow's milk allergy was 43.2%, and predictive factors for egg sensitisation are discussed. 81.8% of the sensitised patients presented with symptoms when exposed to egg in at least one of its forms, although up to 54.5% of patients tolerated boiled egg and egg-based products. Of the non-sensitised patients, the vast majority (92.5%) did not present with symptoms after the introduction of egg in their diet. CONCLUSIONS: Coexistence of allergy to egg and milk allergy is common, and it is recommended that these patients be monitored, since children who are sensitised to egg despite having never been exposed to it in their diet, may present with symptoms immediately following first ingestion. Most children who are initially non-sensitised to egg do not require special care, and it is not generally recommended to delay or monitor these children, although a small number may have subsequently reacted to egg.

Interpretation of a serial Mantoux test taking into account the annual risk of tuberculous infection.

BACKGROUND: Interpretation of tuberculin tests (TSTs) can be difficult. However, it is even more difficult to classify an individual as infected or non-infected if he or she has undergone a prior TST, as the difference between the booster effect and true conversion is not always clear. OBJECTIVE: To determine the size of the induration that is indicative of true infection after a second Mantoux test, based on the fact that the annual risk of tuberculous infection (ARTI) is equivalent, regardless of whether it is calculated directly or indirectly. METHOD: We performed two TSTs in the same population (aged 6 years at the first examination) with a 1-year interval. We calculated the prevalence of infection the first year (1.16%, 95%CI 0.67-1.65) and the ARTI using the indirect method (0.18%, 95%CI 0.00-0.37). For the second year, we considered different induration sizes and finally accepted a diameter of >10 mm as the criterion according to which the ARTI, calculated directly, corresponded to the value of the previous year (0.18%). CONCLUSION: Although they served as an aid in the interpretation of the TST, calculations of this type will probably be unnecessary in the future, when in vitro diagnostic tests for tuberculosis are made available in every health centre, thus enabling the classification of those individuals with indurations of intermediate size in the TST.

[Intracranial hemorrhage due to aneurysms and arteriovenous malformations during pregnancy and puerperium]. / Hemorragia intracraneal por aneurismas y malformaciones arteriovenosas duranteel embarazo y el puerperio.

INTRODUCTION: Subarachnoid hemorrhage for aneurysms and arteriovenous malformations is most frequent during pregnancy and puerperium. We analyzed behavior and management of this patients and the effect on it of our Maternal Health Program. PATIENTS AND METHODS: There were 437 pregnancy and 717 puerperal complicated patients between january 1996 to december 2005, 41 maternal deaths of all causes and 14 indirect maternal deaths. There were operated on 454 patients with intracranial aneurysms and arteriovenous malformations in this period. Pregnant patients and fetus were evaluated to select mode of delivery. A specialized medical team managed precociously these patients. Vascular lesions were clipped by microsurgical methods. Anesthetic management considered pregnancy and fetal complexities. We did not use endovascular methods. Postoperative management was performed on intensive care unit. RESULTS: There were eight pregnant and puerperal patients with subarachnoid hemorrhage due to aneurysms and arteriovenous malformations. 2% of all patients were operated on by this cause. Six harboured intracranial aneurysms and two arteriovenous malformations. We performed seven surgical procedures, five for aneurysms and two for malformations. There was a delivery rate of 2.1 and seven normal newborn. Four patients achieved a complete neurological recovery, two a partial recovery and there were two deceased. This cause constituted 14% of all indirect maternal deaths. CONCLUSION: A precocious clinical diagnosis, neuroimaging studies and interdisciplinary management that involve intensive therapy, obstetric attention and neurovascular surgical treatment determined an increment in the diagnosis of these lesions that require opportune microsurgical or endovascular treatment to prevent maternal death and fetal damage.

[Male pseudohermaphroditism]. / Seudohermafroditismo masculino.

Diagnosis of ambiguous genitalia in a newborn is an emergency that can be difficult to manage, not only because salt wasting entities must be ruled out, but also due to the importance of gender assignment before psychological gender is established. We report two cases of male pseudohermaphroditism, a true hermaphroditism and a 5-alfa-reductase deficiency. The physiology of sexual differentiation and diagnosis, as well as the management of these infants, are discussed.

Cierre progresivo de heridas en pacientes con pie diabético / Progressive suture in diabetic foot patients with difficult healing

Resumen Introducción: Los pacientes diabéticos insulino requirientes con heridas del pie, producto de amputaciones con exposición de huesos, cartílagos o tendones, son de difícil manejo por el déficit de irrigación distal. Alternativa de tratamiento a los colgajos pediculados o microquirúrgicos es el cierre progresivo de las heridas. Objetivo: Reportar la técnica de cierre progresivo de heridas de pie en pacientes diabéticos insulino requirientes. Materiales y Método: Con esta técnica se operó a 15 pacientes diabéticos insulino requirientes que, después de amputación, presentaron heridas del pie con exposición de huesos, cartílagos y tendones. Lesiones de talón: 4, lesiones de antepie: 5 y lesiones plantares: 6. Las edades fluctuaron entre 45 y 70 años. Fueron 11 hombres y 4 mujeres operados entre 2014 y 2019. En ambos bordes de la herida se instaló dos placas de Ventrofil® unidas por sutura en U transversa y se tensó cada 3 días hasta obtener cicatrización. Resultados: 14 pacientes obtuvieron cicatrización en un plazo de entre 15 y 21 días y sólo uno presentó sufrimiento de los bordes de la herida por afrontamiento muy seguido. El seguimiento fue de tres meses sin recidiva de las heridas. Discusión: En pacientes diabéticos las heridas, producto de amputaciones y con exposición de huesos, cartílagos y tendones, son de muy difícil tratamiento debido el déficit de irrigación distal. En estas condiciones el uso de complejos colgajos locales o microquirúrgicos es de alto riesgo dada la posibilidad de necrosis. En esta situación, el cierre progresivo con sutura de Ventrofil® es una alternativa viable, segura, reproducible y con buenos resultados. Conclusión: Es un procedimiento rápido, seguro y reproducible.

Introduction: The closure of wounds in diabetic foot patients, after amputation with esposure of bones, is difficult because of lack of blood supply to this area. The progressive suture with Ventrofil® is a good alternative. Aim: Report the technique of progressive closure of foot wounds in insulin-requiring diabetic patients. Materials and Method: 15 patients with diabetic foot were operated on in the Hospital del Salvador, between 2014 and 2019.The ages were among 45 and 70 years. 11 men and 4 women. In those patients two plaques of Ventrofil® were positioned in both borders of the wound an tied through a horizontal suture.This suture was tensioned every 3 days until get the complete wound healing. Results: 14 patients healed very well and in just only one patient there was suffering of the edges of the wound. The follow up was of 3 months without relapse. Discussion: In patients with diabetic foot with wounds after amputation the closure is difficult because of lack of adecuate blood supply. In these case the progressive suture with plaques of Ventrofil® is a good alternative to a more complex flaps like microsurgicals ones. Conclusion: This technique is sure, easy to perfom and safe.

Pharmacokinetics of cyclosporine as a function of energy--protein deficiency in children with chronic renal failure.

The present study was conducted to determine whether malnutrition in patients with chronic renal failure requiring cyclosporine therapy for renal transplantation has some effect on the clinical pharmacokinetics of cyclosporine. Eleven pediatric patients were enrolled in this study before renal transplantation and divided into two groups (group I: six well-nourished patients with a deficit in weight/height ratio < or = 7%; group II: five malnourished patients with a deficit in weight/height > 8%). The patients received a single oral dose of cyclosporine (3.0 mg/kg). Blood samples were collected for a 26-hour period, and serum concentrations of cyclosporine were measured using fluorescence-polarization immunoassay technology. The results suggest that, when malnutrition is present, the median Cmax of cyclosporine decreases by almost threefold (from 387.5 ng/mL in group I to 136.1 ng/mL in group II). An observed 52% reduction in AUC0-infinity (from 2,856.0 ng/mL/hr in group I to 1,481.4 ng/mL/hr in group II) was caused by the increased volume of distribution (from 4.6 L/kg in group I to 11.1 L/kg in group II). The elimination half-life (t1/2) was longer in group II compared with that of group I (12.4 hr for group II; range, 7.8-13.5 hr versus 8.9 hr for group I; range, 5.2-16.0 hr). Differences in t1/2 were not statistically significant at 5% confidence intervals. The effects of energy malnutrition on the pharmacokinetics of cyclosporine could explain in part some of the interindividual variability. This study provides pharmacokinetic guidelines for the use of cyclosporine.

Addison disease after appendicitis.

A 14-year-old boy manifested acute abdominal pain, vomiting, high temperature and diarrhea. He also underwent increasing hyponatremia and hyperkalemia after appendectomy. Further testing confirmed Addison disease. The serum adrenal antibody test was positive, and other autoimmune diseases were excluded.

[Outcome of tunneled hemodialysis catheters as permanent vascular access]. / Seguimiento a largo plazo de catéteres permanentes en pacientes con dificultad en la obtención de un acceso vascular definitivo.

UNLABELLED: Tunneled cuffed hemodialysis catheters (THC) are developed as a means of short hemodialysis access while a more permanent form of access are maturing. The aim of this study is to investigate the effectiveness, survival and complications of the THC used for long-term vascular access. METHODS: In a retrospective study we looked at 42 THC inserted between November 2000 and October 2003, in 40 elderly patients, with systemic disease or when other vascular access was not possible. RESULTS: Procedural complications occurred in 5 cases (11.9%), which included: local haemorrhage (3), hemothorax (1) and one fatal venous tear. 6 catheters (14.2%) were removed due to complications (non-function 3 and bacteraemia 3). The total incidence of THC related infections was 0.18 episodes/1,000 catheters-days. Patients were followed up for a mean 379 days (range 1-1,140) and a total of 15,946 catheter-days. Qb and KT/V achieved at one month were 278.3 +/- 34.1 ml/min and 1.48 +/- 0.27 respectively. At the end of the follow-up, 23 patients (54.7%) were alive with catheter functioning. One, three and twelve months survival was 90.4%, 73.1% and 59.5% respectively. CONCLUSION: The THC may be a useful alternative permanent vascular access for hemodialysis patients when others vascular access are not possible.

[Meningitis by Listeria in children]. / Meningitis por Listeria en niños.

INTRODUCTION: Listeria monocytogenes is a rare cause of bacterial meningoencephalitis in the non-immunocompromised pediatric population. On occasions, the absence of differential characteristics with other bacteria that cause meningitis delays diagnosis and hence treatment, worsening the prognosis. CASE REPORT: We present a case of a previously healthy, non-immunocompromised teenager who was admitted to hospital with meningitis caused by Listeria. DISCUSSION: We review the literature related to this case, noting the increasing incidence of this microorganism in the etiopathogenesis of meningoencephalitis, reason for which it has to be kept in mind in the differential diagnosis at the time of admission.

Comparison of three prognostic indexes in differentiated thyroid cancer.

We investigated epidemiological data and correlation coefficient among three prognostic indexes (TNM, AGES and DeGroot's) in 82 patients with differentiated thyroid carcinoma (mean age: 41.7 +/- 16.6 years and sex ratio F8:M1). Seventy eight cases (95.1%) had papillary and 4 (4.8%) follicular carcinoma. Surgical complications were: vocal cord paralysis 7 cases (8.4%), hypoparathyroidism 10 cases (12.2%) and both in four cases (4.8%). We did not find surgical complications in patients subjected to main limited procedures. After a follow up of 54.5 +/- 44.5 months one patient was dead due to thyroid cancer (1.2%) and 2 patients had tumor relapse (2.4%). We found positive correlation between TNM and AGES, AGES and DeGroot and DeGroot's and TNM indexes, with concordance between them (k = 0.2281, p = 0.001). In our series, follicular cancer prevalence is lower than literature reports. Our results suggest that TNM, DeGroot's and AGES indexes may have similar prognostic value in differentiated thyroid cancer.

[Hartmann's procedure. Institutional experience with 92 consecutive cases]. / Operación de Hartmann. Experiencia institucional de 92 casos consecutivos.

BACKGROUND: Since it's description in 1923, Hartmann's procedure is widely used for the surgical treatment of acute left colonic complications when preoperative bowel lavage is not feasible and/or there is high risk of anastomotic dehiscence. OBJECTIVE: Analyze the results of Hartmann's operation in the surgical treatment of consecutive patients at a single institution during a 30-month interval. TYPE OF STUDY: Prospective, non-randomized and longitudinal study. MATERIAL AND METHODS: Patients treated with the Hartmann procedure between March 1995 and September 1998. Surgical indication, intraoperative findings, morbidity and mortality were analyzed as well as the rate of reestablishment of bowel continuity and it's morbimortality. RESULTS: Ninety-two patients underwent a Hartmann procedure. The mean patient's age was 60 +/- 25 years (range of 21 to 88 years) and 60% were older than 65 years. An emergency operation was carried out in 91% of the cases. Most of the patients had intra-abdominal sepsis (56%) and benign colonic process (83%). The morbidity rate was 34% and mortality rate 19. During follow-up the bowel continuity was reestablished in 32% of the cases without fatalities. CONCLUSIONS: Hartmann's procedure is a good option for non-elective surgical treatment complicated rectosigmoid pathology. The morbidity and mortality of the operation are highly dependent on the degree of preoperative sepsis and the patient's preexisting condition. The rate of reestablishment of bowel continuity was low probably because of short follow-up.

[Dose-effect of the administration of ferrous fumarate in aged persons with iron deficiency]. / Dosis efecto de la administración de fumarato ferroso en personas ancianas con deficiencia de hierro.

BACKGROUND: Anemia is the most prevalent hematological problem in elderly persons, affecting 14% of the males and 6% of the females of the population over 60 years of age in Mexico City. OBJECTIVE: To determine the effect produced by the prolonged administration of ferrous fumarate in elderly persons with iron deficiency. METHOD: In a population of 178 subjects, aged between 65 to 100 years, iron deficiency was diagnosed in 51 (28.6%), who had serum iron concentrations below 80 micrograms/dL for men and 60 micrograms/dL for women, but only 21 patients (11.8%), accepted to participated in the study. The response to a 6 months oral administration of ferrous fumarate were studied with a daily oral dose of 5 mg/kg of elemental iron. The patients were classified in 3 groups according to the abnormal parameters of iron metabolism (group 1 = 10.9% anemia, group 2 = 28.0% and group 3 = 63.0% anemia). RESULTS: The efficacy of treatment was evaluated by quantification of the changes occurred in serum iron concentrations, hemoglobin, ferritin and transferrin saturation index, at 0, 30, 90 and 180 days of treatment. This study showed that the treatment of oral ferrous fumarate in elderly patients with iron deficiency, produces a quantifiable improvement in measures of iron parameters within 6 months. CONCLUSIONS: The results of this study suggest the usefulness of prolonged treatment with ferrous fumarate in elderly patients with iron deficiency, to avoid therapeutic failure as a consequence of non-compliance as is common in elderly patients.

Post-operative complications following adenotonsillectomy in children with severe sleep apnea-hypopnea syndrome. Do they need to be admitted to an intensive care unit?

INTRODUCTION AND OBJECTIVES: In recent years, with the rise of sleep-disordered breathing, we have been seeing more articles related to post-operative complications after adenotonsillectomy in children with sleep apnea-hypopnea syndrome (OSAS), especially in those with severe sleep apnea. The objective of this study was to evaluate post-operative complications in children with severe OSAS compared to children who had adenotonsillectomy for a different reason, and establish whether they needed admission to an intensive care unit or not. METHODS: All children undergoing adenotonsillectomy in our hospital in the last 5 years were initially included in this study. Complications were analysed with a retrospective review. RESULTS: Two hundred and twenty nine children admitted for adenotonsillectomy were finally included. In the whole group, complications occurred in 3.5% of children, 2.2% corresponding to respiratory complications. Children with sleep apnea (3.23% vs 1.47%, P=.39) or severe sleep apnea (3.77% vs 1.70%, P=.32) presented a higher incidence of respiratory complications, which was not statistically significant and was far below those published by other authors. All respiratory complications took place in the immediate post-operative period (operating theatre or anaesthesia recovery), with none in the paediatric ward. CONCLUSIONS: In our population, children who undergo adenotonsillectomy, without any other comorbidities, malformation syndrome or neuromuscular disease, are more than 2 years old and have an immediate postoperative period without incidence, do not need to be systematically admitted to an intensive care unit, even if they present with severe OSAS.

[Left ventricular mass, forced baseline spirometry and adipocytokine profiles in obese children with and without metabolic syndrome]. / Masa ventricular izquierda, espirometría basal forzada y perfil de adipocitocinas en niños obesos con y sin síndrome metabólico.

INTRODUCTION: Recent reports have shown an increase in changes in cardiac and pulmonary function among obese patients. Furthermore, it has also been demonstrated that obesity is a state of chronic inflammation. We hypothesized that obese children with metabolic syndrome exhibit a higher percentage of left ventricular hypertrophy and altered spirometry values due to higher levels of inflammation. PATIENTS AND METHODS: Left ventricular mass was studied using echocardiography, baseline forced spirometry by spirometer (FlowScreen) and adipocytokine profiles (adiponectin, IL-6, leptin, MCP-1, PCR-Hs, RBP-4, TNF-( and visfatin) were evaluated in peripubertal obese children with and without metabolic syndrome. RESULTS: Forty-one patients (20 girls and 21 boys) were included in the study, 20 of whom (10 boys and 10 girls) were subjects with metabolic syndrome. Of the adipocytokines studied, only leptin, hs-CRP, MCP-1, and the leptin/adiponectin ratio yielded values that were substantially greater in the group with metabolic syndrome (P<.01). An analysis of left ventricular mass index and baseline spirometry showed no differences between the groups studied. However, of the entire cohort, 9.5% had left ventricular hypertrophy. No significant relationship was found between anthropometric data and adipocytokines and the parameters used to study left ventricular mass and spirometry values on the other. CONCLUSION: At the time the study was performed, left ventricular mass and baseline forced spirometry did not appear to be influenced by inflammatory mechanisms.

Qualification and quantification of seventeen natural steroids in plasma by GC-Q-MS and GC-IT-MS/MS.

Studying the plasma steroid profile offers information about the possible existence of endocrinological alterations. This study describes the development and validation of gas chromatographic-mass spectrometric and gas tandem mass spectrometric methods for the simultaneous identification of 17 steroid hormones in human plasma using five different solvents. The n-hexane/ethyl acetate solvent mixture, in a proportion of 70/30 (v/v) provided the best results. The extracts were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide. The obtained limits of detection were below 1 ng/mL in the majority of the studied steroids and the limits of quantification were below 5 ng/mL; the method obtained good linearity, reproducibility, repeatability, accuracy and recoveries above 95% in most cases.

Estandarización de técnicas para el diagnóstico molecular de flavivirus y alfavirus / Standarization of molecular techniques for diagnosis of flavivirus and alphavirus

Los flavivirus y alfavirus afectan la salud de los humanos. En Venezuela y de forma particular en Aragua, las comunidades se han visto afectadas por algunos de los miembros de estos géneros, como Dengue (DENV), y Chikungunya (CHIKV). DENV circula en Aragua desde 1989 causando brotes de importancia clínica, mientras que CHIKV hizo su aparición en 2014. En Aragua, el diagnóstico se había dirigido hacia la detección de DENV, sin embargo la situación con CHIKV generó la necesidad de ampliar el espectro diagnóstico hacia otros agentes virales. En el Laboratorio Regional de Diagnóstico e Investigación del Dengue y otras Enfermedades Virales se adaptaron dos protocolos de RT-PCR previamente descritos para detectar miembros de estos géneros haciendo uso de cepas controles para flavivirus (DENV y Zika) y alfavirus (CHIKV). Ambas técnicas sufrieron modificaciones en la concentración de algunos reactantes (MgCl2, dNTP´s, y cebadores) utilizados en la segunda reacción de PCR. El resto de las condiciones se mantuvieron iguales a las descritas originalmente. Las metodologías estandarizadas permitieron amplificar hasta 1 fg de ARN viral de los controles empleados (Zika y CHIKV) con posibilidad de amplificar cantidades menores a esta. En todos los casos se obtuvieron bandas nítidas e íntegras de acuerdo a los tamaños esperados haciendo uso de cepas previamente identificadas de DENV, CHIKV y Zika. La puesta en marcha de estas metodologías permitirá fortalecer el diagnóstico oportuno de miembros de estos géneros en el marco del sistema de vigilancia epidemiológica de enfermedades virales.

Flavivirus and alphavirus affect the health of humans. In Venezuela and particularly in Aragua, the communities have been affected by some members of these genus, such as dengue (DENV), and Chikungunya (CHIKV). DENV circulating in Aragua since 1989 generating outbreaks of clinical importance, while CHIKV made its appearance in 2014. In Aragua state, the diagnosis had been directed toward the detection of DENV, however the situation with CHIKV generates the need to expand the diagnosis spectrum to other viral agents. In the LARDIDEV we adapted two methodology of RT-PCR´s previously de scribed to detect members of these genus using flavivirus (DENV y Zika) and alphavirus (CHIKV) control strains. Both techniques were modified in the concentration of some reactants (MgCl2, dNTP´s, and primers) in the second reaction of PCR. The other conditions are kept equal to those originally described. Both techniques allowed amplified up to 1 fg of RNA of viral strains (Zika and CHIKV virus), but it could to amplify smaller amounts. In all cases, sharp bands according to the expected sizes using previously identified strains of DENV, CHIKV and Zika virus were obtained. The implementation of these methodologies will strengthen the timely diagnosis of members of this genus within the system of epidemiological surveillance of viral diseases.