Usefulness of PCR strategies for early diagnosis of Chagas' disease reactivation and treatment follow-up in heart transplantation.

Heart transplantation (HTx) is a useful therapy for end-stage Chagas cardiomyopathy; however, Chagas reactivation remains a mayor complication. Parasitological methods offer poor diagnostic sensitivity, and use of more sensitive tools such as the Polymerase chain reaction (PCR) is usually necessary. In the present study, reactivation incidence and PCR usefulness for early reactivation diagnosis, as well as for treatment response evaluation during follow-up, were analyzed using Strout parasite detection test, in 10 of 222 consecutive HTx patients suffering Chagas cardiomyopathy. PCR strategies targeted to minicircle sequences (kDNA, detection limit 1 parasite/ 10 mL blood) and miniexon genes (SL-DNA, 200 parasite/10 mL) were performed to compare parasite burdens between samples. No patients received prophylactic antiprotozoal therapy (benznidazole). Five patients (50%) exhibited clinical reactivation within a mean period of 71.6 days; positive Strout results were observed in most cases presenting clinical manifestations. kDNA-PCR was positive 38-85 days before reactivation, whereas SLDNA-PCR became positive only 7-21 days later, revealing post-HTx parasitic load enhancement present prior to clinical reactivation development. Reactivations were successfully treated with benznidazole and generated negative PCR results. Results observed in this study indicate the value of PCR testing for an early diagnosis of Chagas reactivation as well as for monitoring treatment efficacy.

Albumin nanoparticles for the intravitreal delivery of anticytomegaloviral drugs.

Albumin nanoparticles (NP) were proved to be effective and safe carriers for delivering anticytomegaloviral compounds in the vitreous. NP improved the antiviral activity of both ganciclovir and the phosphodiester oligonucleotide analog to formivirsen. NP appeared to be fusogenic carriers able to target the nucleus of cells. In addition, these drug carriers were well tolerated when administered by the intravitreal route and did not induce autoimmune reactions.

Albumin nanoparticles improved the stability, nuclear accumulation and anticytomegaloviral activity of a phosphodiester oligonucleotide.

The goal of this study was to evaluate the potential of albumin nanoparticles as a delivery system for antisense oligonucleotides. Nanoparticles were prepared by a coacervation process and cross-linkage with glutaraldehyde. Phosphodiester (PO) and phosphorotioate (PS) oligonucleotides were either adsorbed on the surface of nanoparticles (PO-NPA and PS-NPA) or incorporated in the nanoparticle matrix (PO-NPB and PS-NPB). When PO-loaded nanoparticles were incubated with phosphodiesterase, only NPB was able to keep the oligonucleotide hybridization capability for at least 60 min. The antiviral activity was evaluated in MRC-5 fibroblasts infected with human cytomegalovirus at a MOI of 0.0035. Both PO nanoparticle formulations significantly increased the antiviral activity of free PO (P<0.001) and NPB showed slightly higher efficacies than NPA (P<0.05). On the other hand, PS exhibited significant higher activity than free PO (P<0.001), however, no significant differences were found between PS-nanoparticle and PO-nanoparticle formulations. These findings were well correlated with the intracellular distribution observed for fluorescent oligonucleotide-loaded albumin nanoparticles. Even these carriers delayed and decreased the uptake of PO by MRC-5 cells, they finally induced a diffused cytoplasmic distribution and major nuclear accumulation. In summary, albumin nanoparticles partially protected a PO against enzymatic degradation and improved their presence in the nucleus and thus, increased its efficiency.

Bovine serum albumin modified the intracellular distribution and improved the antiviral activity of an oligonucleotide.

In the present work, we describe the ability of bovine serum albumin (BSA) to improve the cytoplasmatic delivery of a phosphodiester oligonucleotide (PO), whose phosphorotioate analogue is ISIS 2922 (Vitravene). The changes in intensity and lambda(max) in fluorescence spectroscopy and the increase in fluorescence anisotropy upon complex formation between the oligonucleotide and the protein (PO-BSA) were used to determine the dissociation constant, Km=6.3 x 10(-8) M. The stoichiometry of the complex is mainly 1:1, although 2 PO per BSA have been detected at high PO/BSA ratios. The complexation did not protect PO against enzymatic degradation by snake venom phosphodiesterase (0.1 mg/mL). Fluorescence microscopy revealed that PO-BSA complexes showed a decreased uptake and modified pattern of intracellular distribution with a rapid nuclear accumulation (rather than vesicular localization in the cytoplasm observed for free oligonucleotide). The effect was only observed over a certain threshold of BSA concentration (higher than found in media supplemented with 10% serum). By this way, the interaction with albumin increased the antiviral activity of the oligonucleotide, tested by a plaque reduction assay in MRC-5 fibroblasts infected with human cytomegalovirus (strain RC 256) at a MOI of 0.0035. At 10 microM PO concentration, free PO decreased virus plaques to 85% of the control, while PO-BSA complexes reduced to 60%, in the same order than ISIS 2922. The phosphorotioate analogue complexed by BSA (ISIS 2922-BSA) showed the highest activity with 45% of the control plaques.

Albumin nanoparticles as carriers for a phosphodiester oligonucleotide.

The goal of this study was the evaluation of albumin nanoparticles as drug delivery systems for antisense oligonucleotides. Bovine serum albumin (BSA) nanoparticles were prepared by a coacervation process. A phosphodiester oligonucleotide was either incorporated into the matrix of the particles by incubation with the albumin prior the coacervation process or adsorbed onto the pre-formed nanoparticles. Incorporated and/or adsorbed oligonucleotide was estimated by capillary electrophoresis and fluorescence spectroscopy. The adsorbed amount of oligonucleotide was dramatically dependent on the pH of the medium. Desorption of the oligonucleotide was also affected by the pH and ionic strength of the medium. This indicated that electrostatic forces play a major role in the interaction between the oligonucleotide and the nanoparticles. When the oligonucleotide was incubated with the albumin prior to nanoparticle formation, the profile of release confirmed that a fraction was incorporated into the matrix and its release was controlled by the albumin degradation. The hybridisation capability of the oligonucleotide in both nanoparticle formulations was retained. However, only the oligonucleotide incorporated into the nanoparticle matrix was protected against enzymatic degradation.

[Prevalence of sleep apnea-hypopnea syndrome among long-haul professional drivers]. / Prevalencia del síndrome de apnea-hipopnea del sueño en conductores profesionales de largo recorrido.

UNLABELLED: Sleep apnea-hypopnea syndrome (SAHS) has been associated with traffic accidents. The aim of this study was to estimate the prevalence of SAHS and analyze risk factors. We studied 163 professional drivers (86.7%) of the 188 employed by 25 participating companies. The subjects completed a questionnaire on SAHS symptoms and risk factors and underwent physical examination and conventional nighttime polysomnographic testing. RESULTS: The prevalence of an apnea-hypopnea index (AHI) ( 5 was 25.2% (95% CI 18.7-32.5) among the drivers. The prevalence of SAHS was 8.6% (95% CI 3.4-12.1). The prevalence increased with age (p = 0.012). Sleepiness while driving or habitual snoring had a sensitivity of 67.5%, specificity of 62.6% and a positive predictive value of 38.6% for detecting SAHS. Logistic regression modelling showed that the risk factors were a body mass index over 29 kg/m2 (OR: 3.56, 95% CI 1.53-8.4) and sleepiness while driving (OR: 3.7, 95% CI: 1.303-10.3). CONCLUSION: These results suggest that detecting SAHS among drivers may be useful for preventing traffic accidents; a questionnaire on SAHS symptoms and objective measures such as polysomnography allow cases to be detected and treated.

Prevalencia del síndrome de apnea-hipopnea del sueño en conductores profesionales de largo recorrido / The prevalence of sleep apnea-hypopnea syndrome among long-haul professional drivers

OBJETIVO: Estimar la prevalencia de SAHS y sus factores de riesgo. MATERIAL Y MÉTODOS: Se ha estudiado a 163 conductores profesionales de los 188 que componen las plantillas de las 25 primeras empresas (86,7 por ciento) estudiadas. Se aplicó un cuestionario sobre síntomas de SAHS, factores de riesgo, examen físico y una polisomnografía nocturna convencional. RESULTADOS: La prevalencia de conductores con índice de apnea-hipopnea (IAH) 5 fue de 25,2 por ciento (IC del 95 por ciento: 18,732,5), con SAHS de 8,6 por ciento (IC del 95 por ciento: 3,4-12,1). Se apreció un incremento de la prevalencia con la edad (p = 0,012).La somnolencia al conducir o el hecho de ser roncador habitual tenían una sensibilidad del 67,5 por ciento, una especificidad del 62,6 por ciento y un valor predictivo positivo del 38,6 por ciento para detectar SAHS. En el análisis de regresión logística, los factores de riesgo fueron el índice de masa corporal (IMC) superior a 29 kg/m2 (OR: 3,56; IC del 95 por ciento: 1,53-8,4) y la somnolencia al conducir (OR: 3,7; IC del 95 por ciento: 1,303-10,3).CONCLUSIÓN: Nuestros resultados sugieren que en empresas de transporte la detección de los conductores con SAHS mediante un cuestionario sobre síntomas de SAHS y una medición objetiva, como la polisomnografía, que permitan detectar los casos para su tratamiento puede ser útil en la prevención de accidentes de tráfico (AU)

Brote epidemiológico de distonía aguda poringestión de drogas de diseño / Epidemiological outbreak of acute dystonia due to designer durg intake

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Ganciclovir-loaded albumin nanoparticles: characterization and in vitro release properties.

Ganciclovir is one of the most widely used antiviral drug for the treatment of cytomegalovirus retinitis. Due to its short half-life in the vitreous, frequent administrations are necessary to maintain the therapeutic levels. In this context, the aim of this study was to characterise and in vitro evaluate the drug release properties of three different formulations of ganciclovir-loaded albumin nanoparticles. These carriers were prepared by a coacervation method and chemical cross-linking with glutaraldehyde. Depending on the step where the drug and/or cross-linking agent were added three different formulations were obtained, named models A, B and C. For model A nanoparticles, ganciclovir was incubated with the just-formed albumin nanoparticles. For the other two types of nanoparticulate formulations, the drug was added to a solution of albumin (model B) and glutaraldehyde (model C) prior to the formation of the carriers by coacervation. In all cases, the size of the different nanoparticulate formulations was comprised between 200 and 400 nm and the yield ranged from 50%, in model A, to 65% in model B. Concerning the ganciclovir loading, model B nanoparticles offered the higher capacity to carry this antiviral drug (around 30 microg ganciclovir/mg nanoparticle). On the contrary, the drug loading calculated for model A nanoparticles was only 14.6 microg/mg. The in vitro release profiles of the nanoparticles showed a biphasic pattern, with an initial and rapid release, followed by a slower step for up 5 days. This burst effect was especially relevant in model A (around 60% in 1 h), followed by model B (40%) and less important in model C (20%). The addition of trypsin to the release medium did not have a significant influence on the release characteristics. However, the release of the drug was increased in acidic or basic mediums, due to the disruption of the covalent binding between ganciclovir and the protein matrix via glutaraldehyde. This strong linkage was also confirmed by TLC experiences. In summary, a first step of incubation between the drug and the protein, prior to the preparation of nanoparticles, enabled us to obtain albumin carriers able to release ganciclovir in a sustained way.

Adherence, side effects and efficacy of stavudine plus lamivudine plus nelfinavir in treatment-experienced HIV-infected patients.

OBJECTIVES: To evaluate adherence, side effects and efficacy of a modality of highly active antiretroviral therapy (HAART) in HIV-infected patients. METHODS: In a cohort, prospective study, 65 previously treated patients received stavudine plus lamivudine plus nelfinavir. Fifty-three participants (81%) had a history of intravenous drug use. Patients were evaluated at 3-month intervals. The association of adherence with demographic variables, hepatitis C virus infection, number of stopped antiretroviral regimens, HIV RNA level, CD4 cell count, and adverse effects to drugs was assessed. RESULTS: After a median follow-up of 12 months, 30 participants (46%) showed adequate adherence in all visits. An association was observed between adherence and female sex: 18 of 47 men (38%) vs. 12 of 18 women (67%) presented adequate adherence in all visits (P=0. 0416). An association was also observed between adherence and low baseline HIV RNA level (P=0.0229). Discontinuation of treatment took place because of refusal to take medication in 11 participants (17%) and because of side effects in seven participants (11%). Undetectable HIV RNA level was achieved in 26 patients (40%) at 3 months and in lower percentages at months 6, 9 and 12. CONCLUSIONS: Overall adherence to the employed HAART regimen was poor. Female sex and low baseline HIV RNA were associated with better adherence. Refusal to take medications and side effects were the main reasons to stop therapy. At 3 months' follow-up, virological efficacy was achieved in 40% of patients.

A randomized, comparative study of lamivudine plus stavudine, with indinavir or nelfinavir, in treatment-experienced HIV-infected patients.

OBJECTIVE: To compare adherence and clinical outcome with two modalities of highly active antiretroviral therapy (HAART), in HIV-infected patients. DESIGN: Randomized, open-label, prospective study. SETTING: Tertiary care centre in Spain. PATIENTS: A total of 112 non-naive HIV-infected patients, recruited from March 1998 through August 1998, were studied. INTERVENTIONS: Triple drug therapy with stavudine and lamivudine, plus indinavir or nelfinavir. MAIN OUTCOME MEASURES: Adherence, side-effects, and immunological, virological, and clinical efficacy of treatment were assessed at 3-month intervals. RESULTS: After a median follow-up of 9 months, 32% of patients in the indinavir group versus 50% of those in the nelfinavir group showed adequate adherence in all clinical appointments (P= 0.0559). Adherence was superior in the nelfinavir group in every visit. After 6 months of treatment 48% of subjects in the indinavir group and 70% of those in the nelfinavir group exhibited adequate adherence (P= 0.0311). After 9 months 35% of patients in the indinavir group and 59% of those in the nelfinavir group showed adequate adherence (P= 0.0291). Side-effects provoked discontinuation of treatment in 34% of patients in the indinavir group and 12% of patients in the nelfinavir group (P= 0.0073). Immunological and virological efficacy were similar in both groups. CONCLUSIONS: Adherence to a HAART regimen with stavudine plus lamivudine plus nelfinavir was superior to a regimen with stavudine plus lamivudine plus indinavir. Side-effects provoked more discontinuation of treatment in the indinavir group than in the nelfinavir group.

Determination of oligonucleotide ISIS 2922 in nanoparticulate delivery systems by capillary zone electrophoresis.

ISIS 2922 is an antisense oligonucleotide with antiviral activity against cytomegalovirus. However, its rapid degradation in biological fluids and its low capacity for diffusion across cell membranes limit its therapeutical use. One possibility to overcome these drawbacks consists of using nanoparticles as drug carriers. The aim of this study was to develop an analytical method for determining the amount of ISIS 2922 loaded into albumin nanoparticles. For this purpose, capillary zone electrophoresis (CZE) was performed on a fused-silica capillary filled with borate buffer (12.5 mM, pH 9.5). Paracetamol was used as an internal standard and a diode-array detection system was set at 270 nm. Under these conditions, the limit of quantitation of ISIS 2922 was 1.27 microg and the precision and accuracy of the method did not exceed 7%. Moreover, the use of paracetamol as internal standard and the quantification by means of a 'corrected area' procedure enabled us to reduce the peak variability and accurately determine the amount of oligonucleotide loaded in the albumin nanoparticles. In summary, this assay is a selective and sensitive CZE method for the accurate quantitation of ISIS 2922 oligonucleotide in albumin nanoparticles.

A study of dental staining among competitive swimmers.

OBJECTIVE: To estimate the prevalence of dental stains (DS) in competitive swimmers and quantify the risk of these stains compared with sportsmen in a non-swimmers group in Castellón, Spain. METHODS: Cross-sectional and case-control designs. Between July 1996 and March 1997, 404 subjects, (171 enrolled in two clubs of competitive swimming and 233 sportsmen from two schools), were examined in order to detect and classify yellowish-brown or dark-brown stains on the facial surface of the eight incisors. Participation rates were 88.6% for swimmers, and 95.7% for sportsmen. Mean of participants' age was 12 years, range 7-22 years. Castellón has three public competition swimming pools, two of which are indoors. Two of the pools used chloride products, and the third bromine for the disinfection of water. The recommended hygiene regulations were adhered to. RESULTS: Prevalence of DS was 60.2% in swimmers and 12.9% in sportsmen (P= 0.0001). Risk factors for DS included: use of competition swimming pools, age, gender, years of competition, daily consumption of coffee, red wine, and iron supplement during the last year. Professional dental cleanliness was a protective factor. In a logistic regression analysis, the use of competition swimming pools maintained a high risk of DS, odds ratio (OR)=9.28; 95% confidence interval (CI) 5.21-16.5, adjusted by the other variables. Amongst swimmers, more than 6 h of training a week increased the risk of these stains (OR=3.51; 95% CI 1.35-9.10). CONCLUSION: The study indicated a high risk of DS in competitive swimmers.

Stavudine, lamivudine and indinavir in drug abusing and non-drug abusing HIV-infected patients: adherence, side effects and efficacy.

OBJECTIVES: To compare adherence and clinical outcome with highly active antiretroviral therapy (HAART) in intravenous drug users (IDUs) and subjects with other HIV risk behaviours (non-IDUs). METHODS: A total of 133 non-naive HIV-infected patients, 95 (71%) IDUs and 38 (29%) non-IDUs received triple drug therapy with stavudine, lamivudine, and indinavir. Adherence, side effects, and immunological and virological efficacy of treatment were assessed every 3 months. RESULTS: During a median follow-up of 12 months, 43 patients (32% of the total) showed adequate adherence in all clinical appointments. Adherence was superior in non-IDUs than in IDUs in every visit, but a significant difference was found only at 6 months, when 22 (58%) non-IDUs versus 37 (39%) IDUs were adherent (P = 0.047). Mildly increased bilirubin was observed in 69 (52%) patients, and renal colic in 34 (26%). No difference in side effects was found between IDUs and non-IDUs. After 6 months of treatment, 35 (43%) participants presented a CD4 cell count increase >100x10(6)/l, and 47 (58%) achieved undetectable HIV RNA (lower limit of detection: 200 copies/ml). CD4 cell count and HIV RNA responses were similar in both groups. CONCLUSIONS: Adherence to the employed HAART regimen was poor. Non-IDUs were more adherent than IDUs, but the difference between both groups was small. Side effects and efficacy were similar in IDUs and non-IDUs.

[Hepatitis A, B, and C in an occupational center for the mentally handicapped]. / Hepatitis A, B y C en un centro ocupacional para disminuidos psíquicos.

BACKGROUND: Estimation of prevalence of serologic markers of viral hepatitis A, B and C in students and staff of an occupational centre in Castellón (Spain). METHODS: Serologic markers of hepatitis A (IgG anti-HAV) and B (HBsAg, anti-HBe, anti-HBc, anti-HBs) were detected by radioimmunoassay (RIA) and serologic markers of hepatitis C (anti-HCV) by immunoradiometric assay (IRMA). Ninety per cent of students (54/60) and 80% of staff (8/10) participated. RESULTS: The prevalence of IgG anti-HVA was 55.6% in students and 75% in staff, increasing with age. Considering persons not vaccinated against hepatitis B, the prevalence of serologic markers hepatitis B was 18.5% in students, two HBsAg and anti-HBe positive, and nobody in staff. Serologic markers hepatitis B was associated with duration of stay institutions for mentally handicapped. None of the center was positive for anti-HCV. CONCLUSIONS: Viral hepatitis prevalences present notable differences. To maintain a serological surveillance of these diseases is important to control and prevention.

A trial comparing nucleoside monotherapy with sequential therapy with 3 drugs in HIV-infected patients.

53 HIV-positive patients, 66% of them zidovudine-experienced, were randomized to receive monotherapy with zidovudine or sequential therapy with zidovudine, didanosine and zalcitabine. Clinical end points, CD4 cell count change, and analysis abnormalities showed better results with sequential therapy.

[Factors associated with sporadic cases of salmonellosis in 1- to 7-year-old children. Study of cases and controls]. / Factores asociados con casos esporádicos de salmonelosis en niños de 1 a 7 años. Estudio de casos y controles.

BACKGROUND: Knowledge about salmonellosis risk factors mainly comes from foodborne outbreaks, and we know little about sporadic cases epidemiology. However most of the cases are sporadic, specially children. This study aims to find out some of determinants of these cases. METHODS: A case-control study with incident cases and controls from the same base population (laboratory diagnosed cases). Cases were children 1-7 years old, affected by diarrhea with culture stools positive to Salmonella between december 1994 and december 1995. Controls from the same source, but positive culture to Campylobacter or viruses. We study food and other environmental risk factors. Odds ratio (OR) are calculated adjusted for age, sex, and year period (cool and cold) by logistic regression. RESULTS: Eating minced meat during three days before symptoms, OR 4.07 (1.20-13.8) and OR 5.63 (1.34-23.6); pets, OR 8.27 (1.96-34.9), and antibiotics the week before symptoms, OR 4.75 (0.84-27.0) were epidemiologically associated with salmonellosis diarrhea. CONCLUSIONS: Epidemiology of salmonellosis sporadic cases in children seems different to the foodborne associated cases and is more complex. Minced meat tree days before symptoms, antibiotics the week before symptoms, and pets could be a risk for this kind of cases. Future studies must also take account of this factors.

[Epidemic outbreak of Salmonella richmond infection in Castellón, Spain]. / Brote epidémico por Salmonella richmond en Castellón, España.

A case-control study was carried out to investigate an outbreak of acute gastroenteritis among a military detachment stationed in a rural area of Castellón, España. The purpose of the study was to determine the causes of the outbreak and develop control measures. Of the 153 men in the detachment, 135 were included in the study. Between 9 and 11 August 1993, 45 cases were reported; the patients' average age was 19.2 +/- 1.5 years. The attack rate was 33.3%. The clinical picture was dominated by the following symptoms: diarrhea (76%), vomiting (67%), nausea (67%), and abdominal pain (28%). The median duration of symptoms was one day, and that of the incubation period was 33 hours. Only one patient required hospitalization and all of them recovered. Salmonella richmond (6.7: and :1.2) was isolated in 5 of the 14 stool cultures performed. An association was also discovered between the illness and consumption of water from an aqueduct that flowed near the camp. A logistic regression model showed that consumption of water from this source remained associated with cases after adjusting for age and the consumption of various foods (odds ratio = 96.5; 95% confidence interval, 11.4-814.4). The risk of suffering from the illness rose with the amount of water consumed (chi 2 trend test = 65.4, P < 0.0001). Chemical and bacteriological analyses of the aqueduct water indicated the presence of fecal contamination. The aqueduct had not been subject to sanitary monitoring, even though the water was used to irrigate agricultural crops. The widespread presence in the environment of species of Salmonella was demonstrated. Health education and microbiological studies of water courses can be of great value in preventing such epidemics.

[Epidemic outbreaks of salmonellosis caused by eating eggs]. / Brotes epidémicos de salmonelosis por consumo de huevos.

BACKGROUND: Investigation of outbreaks of foodborne infections (OFI) by Salmonella associated with the consumption of hens' eggs from a same classification center of Castellón, and determination of origin, extension and to take measures of control and prevention. METHODS: Descriptive and case-control studies, microbiologic analysis of feces, suspicious foods, and eggs. Sampling of eggs in the classification center for estimation the prevalence of Salmonlla. RESULTS: In 1992, 5 OFI were detected, 4 collective (1 school, 2 restaurants, and 1 residence) and 1 at home, by the consumption of food prepared with eggs: fried or boiled eggs, omelette, soufflé, 2 times, and home-made russian salad. Five hundred and forty-five persons were exposed and 364 were studied, with 100 case patients and 16 hospitalized. The range of attack rates was 10.5-87.0%. Samonella enteritidis (3 OFI) and S. typhimurium (3 OFI) were the infectious agents. In February 1993, the prevalence of Salmonella in eggs from the center was 0.26% (4/1.524) (S. enteritidis, 2 isolates, and S. typhimurium, 2 isolate), three on the shell, and one in the yolk. Two farmhouses of the six supplied were infected. CONCLUSIONS: These outbreaks indicate that salmonellosis by egg's consumption are frequent here and investigation of infection sources is necessary.