[Clinical course of cytomegalovirus infection in renal transplant recipients]. / Evolución de la infección por citomegalovirus en transplantados renales.

BACKGROUND: The incidence of cytomegalovirus infection or reactivation is 8 times more frequent in transplant recipients than in the general population. AIM: To evaluate the prevalence and usefulness of different diagnostic techniques for cytomegalovirus infection in renal transplant recipients. PATIENTS AND METHODS: Twenty nine renal transplant recipients were followed for at least five months. Cytomegalovirus infection was assessed by the presence of serum antibodies against the virus using ELISA and viral detection in urine and lymphocytes, using classical viral isolation, shell vial assay, and detection of viral genome by polymerase chain reaction. RESULTS: Prior to transplantation, 23 of 27 patients had IgG type anti cytomegalovirus antibodies. In 40%, IgM type antibodies were detected in some moment of the follow up. Three of these corresponded to seroconversion. Cytomegalovirus was detected in urine in 41% of patients and it was not detected in lymphocytes. Shell vial assay detected the virus in 5 of 13 urine samples and in 1 of 7 lymphocyte samples. Polymerase chain reaction was positive in 12 of the 29 patients. In six patients, an acute rejection was postulated and there was no relation of rejection episodes with viral detection. In two patients, a disease caused by cytomegalovirus was postulated. One of these patients had a seroconversion during follow up. CONCLUSIONS: The prevalence of positive serum indices of cytomegalovirus infection was similar to that reported in the general population. However, the frequency of reactivation and viral disease was lower than that reported elsewhere. The techniques used in this study can be useful to confirm the suspicion of cytomegalovirus disease. However they do not predict the occurrence or evolution of the disease caused by the virus nor viral reactivation in renal transplant recipients.

Polymerase chain reaction for Streptococcus pyogenes used to evaluate an optical immunoassay for the detection of group A streptococci in children with pharyngitis.

BACKGROUND: In evaluations of sensitive rapid tests for group A streptococci such as the optical immunoassay (OIA), some samples are positive by the antigen test but negative by culture. A method is needed for resolving these discrepant results. OBJECTIVE: To develop a PCR-based assay to detect group A streptococci and to use it to establish a reference standard for evaluating an OIA for group A streptococcal antigen. METHODS: A PCR assay that detects a segment of the MF gene of Streptococcus pyogenes was developed for the detection of group A streptococci in throat swabs. Paired swabs were obtained from 200 children with symptomatic pharyngitis and used to perform OIA, agar culture, broth-enhanced culture and PCR. As a reference standard any patient with group A streptococci detected by either culture or PCR was considered to be truly positive. RESULTS: In comparison to agar and broth-enhanced culture procedures, OIA had sensitivities of 82 and 80% and specificities of 87 and 89%, respectively. Eight (44%) of 18 samples that were positive by OIA but negative by culture were positive for group A streptococci by PCR. Compared with the reference standard, sensitivities were OIA 76%, agar culture 79%, broth-enhanced culture 86% and PCR 96%. The specificity of OIA was 92%. CONCLUSIONS: PCR can be used to establish a reference standard for evaluating rapid tests for group A streptococci. With this reference standard OIA was nearly as sensitive as but less specific than agar culture for detection of group A streptococci. Maximum detection requires use of both tests.

[Hemophilia A: analysis of intron 18 and intron 7 of factor VIII gene and their role in a diagnostic strategy for carrier detection in a Chilean population]. / Hemofilia A: análisis del intron 18 e intron 7 del gen del factor VIII y su rol en una estrategia de diagnóstico de portadoras en una población chilena.

Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. A larger number of different mutations in the VIII gene have been identified. Thus, the detection of female carriers, depends upon the analysis of DNA polymorphisms in and near the factor VIII gene. Our aim was to develop a strategy, earlier reported, for carrier testing in families at risk of hemophilia A. In this study, we analyzed the DNA polymorphisms in 26 affected families, with use of the factor VIII intragenic polymorphisms identified by the restriction enzymes BclI and AlwNI, and by differential hybridization with sequence-specific oligonucleotide probes recognizing BclI and AlwNI polymorphism. While the DNA polymorphism detected by BcilI site in intron 18 of the factor VIII gene was informative for 38% families studied, the AlwNI/intron 7 polymorphism provided additional information (4%). The carrier status of the remaining 58% could be determined utilizing the other polymorphisms suggested by strategy. The two polymorphic sites used combined with the other polymorphisms, intragenic and extragenic, can generate levels of informativeness greater than 98%. We concluded that the strategy for carrier testing would be a good alternative in genetic counselling for hemophilia A, but its limitations must be carefully taken into account.

[Activated C protein resistance: laboratory study and prevalence of the defect in the Chilean population]. / Resistencia a la proteina C activada: estudio de laboratorio y prevalencia del defecto en la población chilena.

Activated protein C resistance (APCR) or Factor V Leiden has been recently described as the most prevalent hemostatic abnormality associated with venous thrombosis. In patients with familial thrombophilia, the prevalence of APCR is 19-60% and around 20% in sporadic venous thrombosis. APCR is usually measured by the degree of prolongation of Activated Partial Thromboplastin Time (APTT) on patient's plasma, induced by addition of APC in comparison to normal plasma. At the molecular level the defect is caused by a single-point mutation in the gene for factor V (FV) (G1.691-->A), that predicts the replacement of Arg506 by Glutamine. This mutation makes activated factor V resistant to inactivation by APC. Since the prevalence of the defect is highly variable among different populations, the objective of this work was to study its frequency in our population and in patients with thrombophilia. We defined the normal range for APTT ratio (APTT + APC/APTT - APC) in a group of 73 healthy volunteers in whom the presence of FV Q506 mutation was searched using Mull enzyme digestion of PCR amplified genomic fragment containing the nucleotide 1.691. The lower limit of APTT ratio established in this group was 2.13. APCR was found in 6 out of 159 control subjects (3.8%) and in 14/50 (28%) of patients with thrombosis. In 13 cases as a single defect and in one associated to type I protein C deficiency. All the APCR patients and control subjects were heterozygotes by gene analysis. The results demonstrate that in our population APCR is also the most common defect associated with thrombosis, in accordance with a high prevalence in the population. The ability to screen for this defect will permit the identification of carriers that would benefit of preventive therapy at risk situations.

[Incidence of congenital cytomegalovirus infection in newborn infants of different socioeconomic strata]. / Incidencia de infección congénita por citomegalovirus en recién nacidos de distinta condición socioeconómica.

Cytomegalovirus is the main agent of congenital viral infections. The aim of this study was to compare the incidence of congenital cytomegalovirus infections of two groups of newborns of differing socioeconomic status. Cytomegalovirus was isolated from urine or oropharingeal secretions in 218 children born in a private clinic and 471 born in a public hospital. Positive viral isolates were confirmed with indirect immunofluorescence using monoclonal antibodies. Infection was detected in 12 children (1.82%), four coming from the private clinic (1.86%) and 8 coming from the public hospital (1.81%). Ninety two percent of infected children were asymptomatic. Urine and oropharingeal secretion samples had the same yield for viral isolation. It is concluded that the incidence of congenital cytomegalovirus infection is similar to that described in developed countries.

[Molecular study of 6 episodes of nosocomial infections produced by methicillin resistant Staphylococcus aureus]. / Estudio molecular de 6 episodios de infección intrahospitalaria causados por Staphylococcus aureus meticilino-resistente.

A critical step in any epidemiologic research concerning nosocomial infections is the precise identification of the responsible pathogen. The present work utilized a molecular approach -plasmids identification, restriction length polymorphism DNA analysis, and random amplified polymorphic DNA- for the characterization of 6 nosocomial outbreaks due to 52 strains of methicillin-resistant Staphylococcus aureus (MRSA). In these episodes, the clinic-epidemiologic and phenotypic analysis (antibiotype) pointed to a nosocomial infection. Through molecular analysis it was possible to establish, in a very precise way, clonality due to MRSA strains in 2 of the studied outbreaks; the same type of analysis allowed to eliminate a MRSA clonal origin in the remainder 4 episodes. The antibiogram was not an useful analytic tool due to its poor discriminatory power. Also, through a PCR procedure, it was possible to identify the presence of the gen mecA in every of the 52 MRSA strains studied.

[Antigenic types of circulating rotavirus in children with acute diarrhea in Santiago de Chile]. / Tipos antigénicos de rotavirus circulantes en niños con diarrea aguda en Santiago de Chile.

Protective immunity against rotavirus infection is directed against antigenic epitopes on the outer capsid proteins VP7 and VP4. The aim of this study was to characterize the VP7 and VP4 antigenic types circulating in different hospital areas of Santiago, Chile, over different time periods. Between April 1993 and April 1994 a total of 1206 stool samples were obtained from children consulting for acute no bloody diarrhea in 5 hospitals representative of the 5 major health areas of Santiago. In addition, 256 rotavirus positive samples, obtained from children with acute diarrhea consulting in the north health area of Santiago between 1985-1987 were studied. All samples were processed for rotavirus by an ELISA and all rotavirus positive samples were VP7 typed (types G1-G4) by a monoclonal antibody based ELISA. 50 rotavirus positive samples were selected for VP4 typing by PCR (types P1-P4). A total of 782 rotavirus positive samples were obtained of which 618 (79%) were typable for one specific VP7 type. VP7 type G1 represented 63% of the rotavirus positive samples and predominated in all areas evaluated throughout the entire period of observation. VP7 type G2 represented 13% of rotavirus samples, following G1 in predominance. G2 types decreased progressively in all areas in both study periods. G4 types were detected mainly during 1985-1987, and G3 types have so far not been detected. Preliminary analysis of VP4 types suggests that P1 types are predominant and closely associated with VP7 G1 type. These results are relevant for the adoption of appropriate preventive strategies for rotavirus infection, specifically aimed to the development of effective vaccines.

Enzymatic DNA amplification (PCR) in the diagnosis of extrapulmonary Mycobacterium tuberculosis infection.

A polymerase chain reaction able to amplify Mycobacterium tuberculosis DNA from clinical samples of extra-pulmonary origin is described. The PCR amplified a 294 base pair DNA fragment spanning positions 5'-782 to 3'-1075 of the 65 kDa M. tuberculosis antigen gene. The procedure enables amplification of target DNA at quantities as low as 1 pg of purified material and less than 1000 mycobacteria present in clinical samples. The reaction amplifies M. tuberculosis DNA as well as Mycobacterium bovis BCG DNA. In 34 extra-pulmonary clinical samples studied, 18 rendered positive results and two false-negative results; compared to classical diagnostic procedures, the sensitivity was 90% and specificity 100%. The PCR approach to diagnosis of tuberculosis of extra-pulmonary origin is a valid diagnostic alternative to classical procedures.

[Amplification of 2 different gene segments of Mycobacterium tuberculosis and its usefulness in the diagnosis of tuberculosis]. / Amplificación de 2 segmentos génicos diferentes de Mycobacterium tuberculosis y su utilidad en el diagnóstico de la tuberculosis.

The infection caused by Mycobacterium tuberculosis is highly prevalent in our country and is considered an emergent pathology in developed countries. The amplification of specific gene segments with diagnostics purposes is an alternative to identify fastidious and slow growing infective agents, being Mycobacterium tuberculosis one of them. Two polymerase chain reactions (PCR) directed to the amplification of a 294bp gene segment encoding a portion of a 65KD heat shock protein and 317 bp gene segment of a repetitive DNA segment (IS 6110) of Mycobacterium tuberculosis, have a sensibility of 80 to 91.3% and a specificity of 83.9 to 100% when used in the identification of Mycobacterium tuberculosis in 2 group of clinical samples, both compared to Koch's culture and or Ziehl-Neelsen stain. The diagnostic procedure is particularly useful in diagnosis of tuberculosis of extrapulmonary origin.

In vitro antibacterial activity of trospectomycin (U-63,366F) against anaerobic bacteria and aerobic gram-positive cocci in Chile.

The in vitro activity of trospectomycin sulfate was compared with those of several antimicrobials, against 301 anaerobic bacteria and 613 aerobic Gram-positive cocci. Trospectomycin was about 4- to 32-fold more active than was spectinomycin. Trospectomycin exhibited consistently good activity against all Bacteroides fragilis group isolates, except Bacteroides vulgatus, and against all other anaerobes comparable or higher to that of clindamycin. The trospectomycin's activity was most similar to that of vancomycin, even against methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis.

[The impact of molecular biology in medicine]. / Impacto de la biología molecular en medicina.

Over 30 years ago the genetic material of most organisms was shown to be deoxyribonucleic acid. A considerable amount of information on the fine structure and function of cells has accumulated during this time and important methodological and conceptual advances have occurred. The molecular biology techniques, restricted for many years to basic biologic research, are now being introduced in several areas of clinical medicine. These new ideas are changing the way scientists and physicians think about normal cell function and disease. The DNA recombinant methods allow us to define the molecular mechanisms of several genetic diseases and consider new therapeutic approaches. The biotechnological industry is now producing hormones, peptides and several vaccines by manipulation of genes in bacteria and cell cultures. Our understanding of cell growth and cell differentiation is opening new ways in cancer research; the use of DNA probes in the diagnostic laboratory is exciting to the clinical microbiologist. Molecular biology will continue to advance in the next decades with increasing economic, social and ethical implications.

Estado actual de la sensibilidad in vitro de cocáceas gram positivas aeróbicas / State of in vitro sensibility of gram positive aerobic cocci

Debido a la escasez de publicaciones recientes acerca de las sensibilidades in vitro de cocáceas Gram positivas aeróbicas, hemos considerado de interés analizar estos datos. Del análisis realizado se desprende que los microorganismos de mayor resistencia son los Staphylococcus aureus meticilino-resistentes, para los que el antibiótico adecuado en su manejo clínico debiera ser vancomicina, por sus características de sensibilidad. Los streptococcus faecalis conservan sus niveles de resistencia frente a ampicilina y penicilina. Por último, los Staphylococcus epidermidis no aparecen tan sensibles a las cefalosporinas de 1ª generación como en los datos aparecidos en la literatura extranjera. Se recomienda como alternativa en los Staphylococcus aureus la clindamicina, previa determinación de sensibilidad

In vitro susceptibility to 10 antibiotics of methicillin-resistant (MRSA) Staphylococcus aureus in Chile.

The in vitro activity of 10 antibiotics was determined for 231 strains of methicillin resistant (MRSA) Staphylococcus aureus. Oxacillin was a very good antibiotic to determine methicillin-resistance. Its agreement with methicillin-resistance was in all the strains tested. On the contrary, the correlation with nafcillin was established only in 95% of the strains tested. Cloxacillin and flucloxacillin are not good methicillin-resistance indicators. The strains tested against macrolides, such as erythromycin, and lincosamides, such as lincomycin and clindamycin presented a susceptibility of 68, 78, and 80%, respectively. All tested strains were susceptible to vancomycin.

Biosynthesis of the neurofilament heavy subunit in Xenopus oocytes microinjected with rat brain poly(A)+ RNA.

In order to study the expression of the major subunit of neurofilaments (NFs), rat brain poly(A)+ RNA was purified by three different procedures and was injected in Xenopus laevis oocytes. This system was able to translate efficiently the 200 kDa NF subunit as shown by a dot-blot immunoassay and by immunoprecipitation of labeled NF polypeptides.

ADN recombinante en el diagnóstico clínico / Recombinant DNA in the clinical diagnosis

La biología ha evolucionado al grado que hoy sus métodos de análisis son a nivel molecular. Derivado de este conocimiento se han desarrollado tecnologías altamente sofisticadas; una de ellas es la tecnología de ADN recombinante, la que permite manipular in vitro la información contenida en el genoma de una célula. Una de sus aplicaciones es en el diagnóstico clínico para la detección de agentes patógenos y/o condiciones particulares del material genético que derivan en una patología determinada

Assembly in an in vitro splicing reaction of a mouse insulin messenger RNA precursor into a 60-40S ribonucleoprotein complex.

An SP6/mouse insulin RNA precursor containing two exons and one intron can be spliced in a partially purified nuclear extract isolated from MOPC-315 mouse myeloma cells. We have detected the putative RNA splicing intermediate (intron-3'exon) in a lariat form, the excised intron in a lariat form, and the mRNA spliced product. The in vitro splicing reaction of gel-purified RNA precursors requires ATP and Mg2+ and was accompanied by the formation of a 60-40S ribonucleoprotein complex. The formation of the 60S complex requires ATP. At least two Sm snRNPs containing U1 and U2 RNAs are components of the 60-40S complex. The assemble of those snRNPs occurs early during the splicing reaction and it requires ATP and intron containing pre-mRNAs.

Partial purification and properties of a pre-mRNA splicing activity.

Precursor RNA substrates for splicing reaction were synthesized in vitro from a plasmid DNA in which the early region 2 gene of adenovirus 2 was fused to an efficient bacteriophage promoter (Salmonella phage 6). Pre-mRNA splicing activity from nuclear extracts of MOPC-315 mouse myeloma cells was partially purified 108-fold by three chromatographic steps. The in vitro splicing reaction catalyzed by the partially purified fractions was efficient (60-80% substrate conversion) and accurate at the nucleotide level. The reaction occurred with crude or purified fractions without any detectable lag and nucleotides (ATP or GTP) were absolutely required. Monoclonal anti-Sm antibodies that quantitatively immunoprecipitate U1 small nuclear ribonucleoprotein particles totally inhibited the splicing activity of the purified fractions, indicating that U1 small nuclear RNPs had co-purified with the activity and were absolutely required for the splicing reaction.