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.

Background: The incidence of cytomegalovirus infection or reactivation is 8 times more frequent in transplant recipients than ain 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 followup. 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.

We have studied two groups of pregnant women 939 adults and 246 adolescents whose first prenatal visit was prior to 14 weeks of gestation. These women belong to a low socioeconomic group attending public obstetric outpatient services of the National Health System (NHS) of Santiago-Chile and the Adolescent Reproductive Medicine Center Medical School University of Chile. The seroprevalence of Cytomegalovirus (CMV) infection was established in the first trimester through an enzymelinked immunosorbent assay test being quite high in both groups: 95% in adults and 89.9% in adolescents. All seronegative women were followed prospectively to document the occurrence of primary CMV infection during pregnancy and the presence of congenital infection in their newborns. We found that 4 of 25 seronegative adolescents acquired CMV during pregnancy as compared to 1 of 47 adult women. Of the babies born to the 4 adolescent mothers two had congenital infections one was not evaluated and the remaining baby had neither clinical nor laboratory findings consistent with CMV as was the case with the child born to the adult mother with primary CMV infection. (excerpt)

Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. A large 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 hemophilic 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 polymorphisms. 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 the 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 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.

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.

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.

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.

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.

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.

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

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 294 bp 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,

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.

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.

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.

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.