Abstract: Introduction: The main role of Vitamin D is to regulate calcium metabolism, whose main source is vitamin D3 ob tained mostly from the action of ultraviolet (UV) light on the skin. Objective: To evaluate the seaso nal differences in the concentrations of 25-hydroxy-vitamin D3 (25OHVitD3), parathyroid hormone (PTH), alkaline phosphatase (ALP), and calcium in school-age children. Subjects and Method: The concentrations of 25OHVitD3, PTH, ALP, and calcium were measured in children from Santiago, Chile (latitude -33.4372), aged 5 to 8 years, without Vitamin D supplementation, in different seasons of the year. VitD status was defined as sufficient with concentrations of 25OHVitD3 >20 ng/mL (50 nmol/L), insufficient 12-20 ng/mL (30-50 nmol/L) and deficient <12 ng/mL (30 nmol/L) based on the recommendations of the expert group of the “Global Consensus for the Prevention and Mana gement of Nutritional Rickets”. Results: 133 children participated (89 preterms under or equal to 32 weeks), 41 during summer, 28 in fall, 35 in winter, and 29 in spring. The difference of means between summer and winter was 9.6 ng/mL for 25OHVitD3 (p <0.0001), -11.1 pg/mL for PTH (p <0.0001), and -47.5 IU/mL for ALP (p= 0.01). There were no differences in calcium concentrations. In sum mer, 97.6% of the subjects were classified with sufficiency status (> 20 ng/mL), which decreased significantly in winter to 54.3% (p <0.0001). Conclusions: In winter, 25OHVitD3 concentrations decreased in approximately half of the children, which was associated with an increase in PTH and ALP, and normal calcium concentrations. According to our results, children may need VitD supple mentation during fall and winter.

Resumen: Introducción: El principal rol de la vitamina D es la regulación del metabolismo del calcio, cuya principal fuen te es la vitamina D3 que se obtiene principalmente por la acción de la luz ultravioleta (UV) en la piel. Objetivo: Evaluar las diferencias estacionales en las concentraciones de 25-hidroxi-vitamina D3 (25OHVitD3), hormona paratiroidea (PTH), fosfatasa alcalina (FA) y calcio en niños en edad esco lar. Sujetos y Método: Se midieron las concentraciones de 25OHVitD3, PTH, FA y calcio en niños de 5 a 8 años, sin suplementación de Vitamina D, reclutados en Santiago de Chile (latitud -33.4372) en distintas estaciones del año. El estatus de VitD fue definido como suficiente con concentraciones de 25OHVitD3 > 20 ng/mL (50 nmol/L), insuficiente 12-20 ng/mL (30-50 nmol/L) y deficiente < 12 ng/ mL (30 nmol/L) en base a las recomendaciones del grupo de expertos del “Consenso Global para la Prevención y Manejo del Raquitismo Nutricional”. Resultados: En total participaron 133 niños (89 prematuros menor o igual a 32 semanas), 41 durante el verano, 28 en otoño, 35 en invierno y 29 en primavera. La diferencia de las medias entre el verano y el invierno fue de 9,6 ng/mL para 25OHVitD3 (p < 0,0001), -11,1 pg/mL para PTH (p < 0,0001) y -47,5 UI/mL para FA (p = 0,01). En las concentraciones de calcio no se observaron diferencias. El 97,6% de los sujetos fueron clasificados con estatus de suficiencia (> 20 ng/mL) en verano, lo que disminuyó significativamente en invierno (54,3%, p < 0,0001). Conclusiones: Las concentraciones de 25OHVitD3 disminuyeron en aproximadamente la mitad de los niños durante el invierno, lo que se vio acompañado de un aumento de la PTH y FA, asociado a concentraciones normales de calcio. De acuerdo a nuestros resultados, la suplementación con VitD en niños podría ser necesaria durante otoño e invierno.

Maturity-Onset Diabetes of the Young (MODY) refers to a heterogeneous group of monogenic diabetes. Unlike other types of MODY characterized by genetic defects in transcription factors, MODY 2 is triggered by metabolic alterations caused by mutations of glucokinase (GCK), the first enzyme of the glycolytic pathway. We report a three-generation Chilean family with multiple cases affected with this disease. The index case is a patient who presented severe neonatal hyperglycemia (831 mg/dl, without ketosis) requiring continuous infusion of insulin, which was suspended after 48 hours with normalization of blood glucose. Subsequently, continuous glucose monitoring at 4 months of age revealed 47% of tissue glucose levels above 140 mg/dl, with fasting glucose levels between 120 and 166 mg/dl. The genetic analysis revealed a previously reported mutation in heterozygous state of the GCK gene (c.148C>T; p.His50Tyr). This mutation was also identified in more than one affected relative in the last two generations, with a transmission pattern suggestive of dominant inheritance. GCK gene sequencing led to a correct molecular diagnosis of MODY 2 while bioinformatic analysis indicated the possible molecular causes of the enzyme dysfunction. The knowledge of the molecular diagnosis allowed an adequate medical treatment for this disease.

We report an asymptomatic 23-year-old woman with an isolated and persistent increase in serum levels of aspartate aminotransferase (AST). An extensive work up including laboratory and image testing revealed no abnormalities thus suggesting the presence of macro-AST. A polyethylene glycol (PEG) precipitation assay was performed and confirmed the presence of macro-AST.

Background: The dose of oral anticoagulants (OAC) shows great variability among patients. Pharmacogenetic studies have shown that common variants in genes CYP2C9 (*2 and *3) and VKORC1 (-1639G>A) are associated with lower requirements of OAC. Aim: To study the association between average maintenance doses of oral anticoagulant therapy required to maintain a stable INR and CYP2C9 and VKORC1 gene variants in Chilean adults. Material and Methods: Prospective study of patients on anticoagulant treatment and with a stable international normalized ratio (INR) for prothrombin time for at least three months. Patients were classified as having high or low acenocoumarol or warfarin requirements. Peripheral blood DNA genotyping was performed by polymerase chain reaction and restriction fragment polymorphism or sequencing and electrophoresis. Results: The study included 185 patients, 125 on acenocoumarol and 60 on warfarin. Patients with VKORC1-1639A allele were more likely to require lower doses of both drugs than patients with the G allele (Odds ratio [OR] for acenocoumarol 9.06, and OR for warfarin = 18.7). There was no association between CYP2C9*2 and*3 and acenocoumarol or warfarin requirements. Conclusions: There is an association between VKORC1-1639A variant and anticoagulant doses.

Background: Molecular techniques for human papillomavirus (HPV) detection have a good performance as screening tests and could be included in cervical cancer early detection programs. We conducted a population-based trial comparing HPV detection and Papanicolaou as primary screening tests, in a public health service in Santiago, Chile. Aim: To describe the experience of implementing this new molecular test and present the main results of the study. Material and Methods: Women aged 25 to 64 enrolled in three public health centers were invited to participate. In all women, samples were collected for Papanicolaou and HPV DNA testing, and naked-eye visual inspection of the cervix with acetic acid was performed. Women with any positive screening test were referred to the local area hospital for diagnostic confirmation with colposcopy and biopsy of suspicious lesions. Results: Screening results were obtained for 8265 women, of whom 931 (11.3%) were positive to any test. The prevalence of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was 1.1%; nine women had invasive cervical cancer. Sensitivities for the detection of CIN2+ were 22.1% (95% confidence interval (CI) 16.4-29.2) for Papanicolaou and 92.7% (95% CI 84.4-96.8) for HPV testing; specificities were 98.9% (95% CI 98.7-99.0) and 92.0% (95% CI 91.4-92.6) respectively. Conclusion: This experience showed that the implementation of a molecular test for cervical cancer screening is not a major challenge in Chile: it was well accepted by both the health team and the participants, and it may improve the effectiveness of the screening program.

Syphilis is a sexually transmitted disease caused by Treponema pallidum. The diagnosis is based mainly in clinical presentation and non-specific assays. PCR-based diagnosis has been suggested as an attractive alternative method. The aim of this study was the validation of a PCR-based test for the diagnosis of early syphilis (ES) and neurosyphilis (NS). Clinical samples of mucocutaneous lesions and cerebrospinal fluid (CSF) specimens from patients previously diagnosed for ES and NS respectively using an enlarged gold standard, were tested by PCR. The reaction was done using primers targeting the tpN47gene. Twenty out of 21 mucocutaneous samples from patients diagnosed with ES were positive by PCR, with a clinical sensitivity of 95%. Four out of 8 CSF samples from patients previously diagnosed with NS were positive by PCR, with a clinical sensitivity of 50%. The clinical specificity for both ES and NS was 100%. The PCR sensitivity and specificity for mucocutaneous samples allowed us to implement this assay in our laboratory for routine diagnosis. Although the sensitivity of the PCR in CSF was low, it may be useful to support clinical diagnosis.

La sífilis es una enfermedad de transmisión sexual producida por Treponema pallidum, cuyo diagnóstico se realiza presuntivamente basándose en aspectos clínicos y análisis de especificidad limitada. La reacción de la polimerasa en cadena (RPC) ha sido planteada como una alternativa diagnóstica de mayor sensibilidad y especificidad. El objetivo de este trabajo fue validar una RPC para el diagnóstico de sífilis temprana (ST) y neurosífilis (NS). Se utilizaron muestras de lesiones muco-cutáneas y de LCR de pacientes con sospecha de cursar ST y NS respectivamente, previamente diagnosticados, utilizando un estándar de oro ampliado. La RPC fue realizada con partidores dirigidos al gen tpN47. De las 21 muestras de pacientes con ST, la RPC resultó positiva en 20, lo que resulta en una sensibilidad clínica de 95%. De las 8 muestras de pacientes con NS, la RPC resultó positiva en 4, obteniéndose una sensibilidad clínica de 50%. La especificidad clínica para ST y NS fue de 100%. La excelente sensibilidad y especificidad de la RPC para muestras muco-cutáneas permitió la exitosa implementación de este análisis en nuestro laboratorio para el diagnóstico de rutina. Si bien la sensibilidad de la RPC en LCR es baja, es muy útil para apoyar el diagnóstico clínico.

Invasive candidiasis (IC) epidemiology has changed in critically ill patients and limited data are available in Chile. Objective: To describe the epidemiological and microbiological profile of IC in critically ill patients. Methods: Observational prospective study conducted from October 2001 to August 2003 in critically ill adults with suspected or confirmed IC. Results: 53 patients met criteria for IC, finding 18 (33.9%) candidemias, 22 (41.5%) disseminated IC, and 13 (24.5%) local IC. We identified 8 (44.4%) C. albicans and 10 (55.6%) non-albicans Candida in candidemias. C. tropicalis was the predominant non-albicans species (27.7%). An 88.8 % of Candidas sp recovered in candidemias were fluconazole susceptible. Overall hospital mortality was 24.5%. Mortality in candidemia was significantly lower than in disseminated IC (16.6 vs 31.8%, p = 0.02). Conclusions: A higher proportion of non-albicans Candida was observed in candidemias from critically ill patients. However, most of these strains were fluconazole susceptible. A lower overall mortality was observed in candidemias.

La epidemiología de candidiasis invasora (CI) ha cambiado, lo cual no ha sido suficientemente estudiado en Chile. Objetivo: Describir el perfil epidemiológico y microbiológico de CI en pacientes críticos. Métodos: Estudio observacional prospectivo entre octubre 2001 y agosto 2003, en pacientes críticos adultos con sospecha o confimnación de CI. Resultados: 53 pacientes cumplieron criterios de CI. De ellos, 18 (33,9%) tuvieron candidemia, 22(41,5%) CI diseminada y 13(24,5%) CI local. Entre las candidemias, hubo 8 C. albicans (44,4%) y 10 Candida no albicans (55,6%), predominando C. tropicalis (27,7%). Un 88,8% de las candidemias fueron susceptibles a fluconazol. La mortalidad hospitalaria global fue 24,5%, significativamente menor en pacientes con candidemias vs CI diseminada (16,6 vs 31,8%, p = 0,02). Conclusiones: Se observó una mayor proporción de Candida no albicans en candidemias de pacientes críticos. Sin embargo, la mayoría de estas cepas fue susceptible a fluconazol. La mortalidad global fue menor en candidemias.

Nowadays, the analysis of genetic markers is a very important and validated tool for the identification of individuals, and for paternity testing. To do so, highly variable regions of the human genome are analyzed, making it possible to obtain the genetic profle of an individual, and to distinguish between different individuals. The methodology used is basically the same all over the world, consisting in the analysis of 13 to 15 markers. To assign biological paternity the child must have inherited the characteristics from the alleged father in each of the genetic markers analyzed. This analysis achieves a certainty higher than with any other test, which is expressed as the probability of paternity. This probability has to be at least 99.9%, but greater probabilities are usually obtained, especially if the mother is included in the analysis. If the characteristics of two or more genetic markers from the alleged father are absent in the child, biological paternity is excluded.

El análisis de marcadores genéticos se ha convertido en una herramienta muy importante y ampliamente reconocida para la identificación de individuos y para el estudio de paternidad. Para esto se estudian distintas regiones del genoma humano que son altamente variables en la población y que permiten obtener el perfil genético y distinguir entre distintos individuos. La metodología que se utiliza es básicamente la misma en todo el mundo y consiste en el análisis de entre 13 a 15 marcadores. La paternidad biológica se asigna cuando el hijo/a presenta las características que debe heredar del presunto padre en cada uno de los marcadores genéticos estudiados. A través de este análisis es posible asignar paternidad con un grado de certeza más alto que con cualquier otro sistema, el que se expresa como probabilidad de paternidad. Esta probabilidad debe alcanzar al menos 99,9%. Sin embargo, es posible obtener probabilidades mucho más altas, sobre todo si se incluye a la madre en el estudio. Si las características genéticas del supuesto padre están ausentes en el hijo/a en al menos dos marcadores, se excluye la paternidad biológica.

The aim of this study was to evaluate the performance of enzymatic hydrolysis of acid or alkali pretreated sugarcane bagasse for the production of fermentable sugars. The first step consisted of selection of commercial enzymes presenting the highest cellulolytic activities. After selection of four enzymes: HPL, CL, P1 and P4, their performances were tested in the bagasse pretreated with acid and alkali. The sugar content of the hydrolysates was analyzed by anion exchange liquid chromatography. Data showed that the joint action of 0.5% acid pretreatment, 121ºC, 30 minutes and enzyme CL provides the best results, 67.25 g of hexose and 148.13g of pentose per kg of dry bagasse.

Background: Hemophilia A is an inherited disorder caused by alterations in factor VIII gene (F8) located on the X-chromosome, the intron 22 inversion being the most common mutation. The rest are predominantly point mutations distributed along this large gene of 26 exons. Aim: To implement a molecular diagnostic test to detect mutations in the F8 gene in Chilean patients with Hemophilia A. Material and Methods: To validate the testing methods, we analyzed samples with intron 22 and intron 1 inversion, and with point mutations previously studied, as well as one subject without Hemophilia. We also studied unrelated Chilean patients with Hemophilia A and their female relatives for carrier testing. Intron 22 and intron 1 inversions were studied by long distance polymerase chain reaction (PCR) and point mutations by sequencing the coding and promoter regions of the F8 gene. Results: The results obtained in all samples used for validation were concordant with those obtained previously. In the Chilean patients, the intron 22 inversion and point mutations previously described were observed. In 6 out of 9 patients with mild Hemophilia A we found the same mutation (Arg2159Cys) in exon 23, which has been described as prevalent in mild Hemophilia A. Conclusions: The analysis of intron 22 and intron 1 inversions, as well as of point mutations in the F8 gene will help us to confirm the diagnosis in patients with severe, moderate and mild Hemophilia A, and also it will allow us to perform carrier testing and to provide better genetic counseling.

It has been demonstrated that HLA-B*5701 screening reduces the risk for hypersensitivity reaction to abacavir in HIV-infected patients. Since B*5701 prevalence varies among different populations, it is important to determine the carrier frequency prior to its use for the screening of HIV-infected patients.The aim of this study was to determine HLA-B*5701 carrier frequency in Chilean general population and HIV-infected patients referred for B*5701 typing. For that purpose 300 blood bank donors and 492 abacavir-naïve HIV-infected patients from Chile were screened for B*5701 by a sequence specific primer PCR.We detected 14/300 (4.7%) B*57-positive individuals in the Chilean general population, 11 (3.7%) were B*5701 positive, and 3 (1%) had another subtype.All were heterozygous,thus a B*5701 allele frequency of 2% was determined.Eleven of 492 (2.2 %) HIV-patients carried a B*5701 allele. The difference between these frequencies is probably due to slow progression of HIV infection in HLA-B*5701 carriers, thus less patients would require antiretroviral therapy and B*5701 typing. Considering the usefulness of B*5701 screening, its prevalence in the Chilean general population,and the availability of a validated method,we conclude that HLA-B*5701 typing in Chilean HIV-infected patients about to initiate abacavir treatment is strongly recommended

The knowledge of the human genome has led to an explosion of available genetic tests for clinical use. The methodologies used in these tests vary widely, allowing the study from chromosomes to the analysis of a single nucleotide. Prior to its use in the clinical setting, these tests should have an evaluation that includes analytical and clinical validation and determination of the clinical utility, as any other tests, including requirements for quality assurance. Recently, the CDC (Centers for Disease Control and Prevention, USA) published a guideline for Good Laboratory Practices for Molecular Genetic Testing for Heritable Diseases and Conditions, covering the pre-analytical, analytical and post-analytical phases of the tests. The document covers the importance of proper selection of tests, the availability of information on the performance of the techniques used, the quality control practices, the training of personnel involved and the report of results, to allow the adequate interpretation, including sensitivity and specificity. Considering that recent advances in genetics have changed and will continue to affect clinical practice, genetic tests must meet quality and safety requirements to enable optimal use of them.

El conocimiento del genoma humano ha dado lugar a un aumento explosivo de los test genéticos disponibles para uso clínico. Las metodologías utilizadas en este tipo de tests son muy variadas, permitiendo desde el estudio de los cromosomas hasta el análisis de una base nucleotídica. Previo a su utilización en el ámbito clínico, estos tests deben tener una evaluación que incluya su validación analítica y clínica y determinación de la utilidad clínica, además de cumplir, como cualquier otro examen, con requisitos para el aseguramiento de la calidad. Recientemente, el CDC (Centersfor Disease Control and Prevention, EE. UU) hapublicado recomendaciones para las buenas prácticas de laboratorio de tests moleculares que se utilizan para el diagnóstico de enfermedades genéticas, que abarcan la fase pre- analítica, analítica y post-analítica. Dentro de éstas destacan: la importancia de la selección adecuada de los tests, la disponibilidad de la información sobre el desempeño de las técnicas utilizadas, las prácticas de control de calidad, la capacitación del personal involucrado y la elaboración de un informe de resultados que permita al clínico interpretarlos adecuadamente, incluyendo sensibilidad y especificidad. Tomando en cuenta que los recientes avances en genética han modificado y seguirán modificando la práctica clínica, los test genéticos deben cumplir con las exigencias de calidady seguridad que permitan su uso óptimo.

Background: Commercial polymerase chain reaction (PCR) kits are widely accepted for analysis of smear positive respiratory specimens, but the sensitivity is variable for smear negative ones. Objective: To assess the PCR method usefulness in smear negative respiratory and non respiratory specimens. Methods: We compared the PCR results (AMPLICOR MTB test™, Roche) of 235 specimens subjected to culture in Loewenstein-Jensen agar (as the gold standard). Results: 181 (76%) were respiratory and 54 (24%) extra-respiratory specimens. The sensitivity was 88%) and 50%>, respectively, specificity and PPV was 100%> in both cases. NPV was 99.4%> in respiratory specimens and 96.1% in non-respiratory specimens. Conclusions: The good performance of this PCR in smear negative respiratory specimens allows the clinician to take decisions based on the result of this exam. In extra-respiratory specimens the contribution is important only when the PCR result is positive.

Introducción: Los métodos comerciales de reacción de polimerasa en cadena (RPC) están validados para el diagnóstico de Mycobacterium tuberculosis sólo en muestras respiratorias con baciloscopia positiva. En muestras con baciloscopia negativa, la sensibilidad es variable. Objetivo: Evaluar el comportamiento de la RPC en uso en muestras con baciloscopia negativa. Método: Se compararon los resultados de 235 muestras analizadas por cultivo en agar Loewenstein-Jensen ("estándar de oro") y RPC (AMPLICOR MTB test®, Roche). Resultados: Se analizaron 181 muestras respiratorias y 54 muestras extra-respiratorias. La sensibilidad de la RPC fue de 88 y 50%), respectivamente, la especificidad y el VPP fueron, en ambos casos, de 100%o, y el VPN fue de 99,4%o en muestras respiratorias y de 96,1%) en extra-respiratorias. Conclusiones: El buen desempeño de esta RPC en muestras respiratorias con baciloscopia negativa permite al médico clínico tomar decisiones en base al resultado de este examen, fundamentadamente. En muestras extra-respiratorias, sólo pueden sacarse conclusiones frente a resultados de RPC positivos.

Background: An alert value is a result suggesting that the patient is at imminent danger unless appropriate remedial actions begin promptly. Report of alert values (AV) by the clinical laboratories has taken special relevance in recent years due to its contribution to patient's care. Aim: To report results of AV informed during 2007 within the Health Network of the Pontificia Universidad Católica de Chile. Material and methods: Analysis of AV recorded in a centralized database of the laboratories of the health network, between January and December, 2007. Results: Total number of AV was 5.366, which represented 0.3% of total examinations and corresponded mainly to the clinical chemistry area. Potassium levels generated the higher number of AV detected, followed by positive blood cultures. Eighty two percent of AV corresponded to hospitalized patients. The greater number of AV was reported to intermediate and intensive care services. Thirty two percent of AV was informed to the physician or professional in charge of the patient within 5 minutes of obtaining the results and 79% within 30 minutes. Conclusions: To obtain a real impact on patient management, it is fundamental to shorten the ¡apse between the obtainment of tests results and the warning, supported on appropriate computerized systems, and to spread the procedure to all personnel involved in patient's care (RevMéd Chile 2009; 137: 1137-44).

The use of techniques for the detection of nucleic acids such as the polymerase chain reaction (PCR) has had a major impact on microbiological analysis, playing an important role in the clinical laboratory. Most of the techniques currently used are designed for specific detection of a particular microorganism. However, infectious agents can also be identified even if genus or species are unknown, using universal primers to amplify bacterial or fungal DNA and then identify the species by sequency (universal or wide spectrum PCR). This methodology is applied in cultures that are difficult to identify usingphenotypic techniques, and more recently it is also being used directly in clinical samples, where the detection and identification of the infectious agent by traditional techniques is difficult or not possible.

El uso de técnicas para la detección de ácidos nucleicos como la reacción en cadena de la polimerasa (PCR) ha tenido un gran impacto en el diagnóstico microbiológico, ocupando un lugar importante en el laboratorio clínico. La mayoría de ¡as técnicas en uso han sido diseñadas para la detección específica de un microorganismo. Sin embargo, también es posible identificar el agente etiológico aunque se desconozca la especie o el género, utilizando partidores universales para amplificar el ADN de bacterias y hongos, y luego secuenciar para identificar la especie (PCR universal o de amplio espectro). Esta metodología se aplica en cultivos difíciles de clasificar por técnicas fenotípicas, pero también se ha comenzado a utilizar directamente en muestras clínicas, en las que la detección e identificación del agente infeccioso por técnicas tradicionales resulta difícil o no es posible.