Mannose-binding lectin exon 1 and promoter polymorphisms in tuberculosis disease in a Mediterranean area.

Mannose-binding lectin (MBL) is a serum protein that activates the complement and mediates phagocytosis. MBL levels and MBL2 genotype may impact upon host susceptibility to tuberculosis (TB) disease but evidence to date has been conflicting. MBL2 exon 1 and promoter genotyping and serum MBL concentrations were determined in 79 patients with active tuberculosis (58 pulmonary TB and 21 extrapulmonary or miliary TB) and 120 household healthy contacts (HHC) from a Mediterranean area (Majorca Island, Spain). Significantly higher serum MBL levels were found in patients with active tuberculosis than in HHC [median MBL concentrations 3430 ng mL(-1) (10-28 415) and 2600 ng mL(-1) (5-20 000) respectively, P = 0.002]. These higher MBL levels were mainly related to the most prevalent YA/YA wild-type diplotype. There was a strong correlation between MBL2 exon 1 and promoter genotype and MBL levels. The diplotype LYQA/HYPA was present in 12 out of 57 of the pulmonary TB cases but in none of the extrapulmonary TB patients. Diplotype LXPA/HYPA, producer of high levels of MBL, was significantly more frequent in HHC than in patients (16.8% vs. 6.4%, P = 0.031) suggesting a protective role against the development of TB disease that has not been previously found.

Molecular epidemiological typing of Enterobacter cloacae isolates from a neonatal intensive care unit: three-year prospective study.

Since 1992, there has been an increase in the incidence of Enterobacter sepsis in the neonatal intensive care unit (NICU) of the authors' hospital. From 1995 to 1997, a prospective molecular epidemiological survey of the colonizing and infecting strains isolated from neonates was conducted. Enterobacter cloacae was the most frequent cause of neonatal sepsis, accounting for 19.2% of all neonatal infections, reaching a peak incidence of 2.2/1000 during 1996. Fifty isolates from the NICU and four epidemiologically unrelated strains were characterized by pulse-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC)-PCR and plasmid profiling. PFGE was the most discriminatory technique and identified 13 types (two of them classified into two and three subtypes) compared with ERIC-PCR, plasmid profiling and ribotyping that identified 11, 11 and seven types, respectively. A good correlation was found between all techniques. Five different clones caused 15 cases of sepsis. Clones A and B were prevalent in 1995 and 1996, but they were not isolated in 1997. An outbreak caused by clone G in 1997 was controlled by cohort nursing and hygienic measures, without changing the antibiotic policy. Strains were characterized by their antibiotic resistance pattern and divided into three groups. Group I correlated with PFGE types A, B1 and B2, which hyperproduced Bush type 1 chromosomal beta-lactamase and expressed extended-spectrum ?-lactamases (ESBLs). Group II only hyperproduced Bush type 1 chromosomal beta-lactamase and correlated with PFGE-types D1, D2, D3 and I. Finally, Group III, with inducible beta-lactamases, correlated with the rest of PFGE types. The sudden disappearance of E. cloacae after reinforcement of hygienic measures confirms the importance of patient-to-patient transmission.

Shell-vial culture and pp65 antigenemia assay in the detection of cytomegalovirus in the first blood sample of renal transplant recipients.

The aim of the study was to compare the efficacy of pp65 antigenemia assay and the shell-vial culture (SVC; viremia) for the diagnosis of cytomegalovirus (CMV) infection in renal transplant recipients, comparing the results obtained in the first blood sample and the total number of blood samples analyzed in this group of patients. During the study period, 70 renal transplant recipients were studied: 44 (62.8%) with CMV infection. The method of sedimentation in a dextran solution for leukocyte extraction was used in the pp65 antigenemia assay. The MRC-5 shell-vial assay was used for CMV isolation from leukocytes (viremia). Eighty blood samples were examined from 70 renal transplant recipients: Of the 44 positive samples studied, in 77.5% of cases, both the antigenemia assay and the SVC were positive. In 16.2%, only the antigenemia assay was positive, and, in 6.2%, only the SVC was positive. In all blood samples studied, the antigenemia was present in 93.7% of cases, and the SVC was present in 83.7% (P = 0.04). If the results obtained in only the first blood sample taken for the diagnosis are studied, then we observe that the antigenemia assay was positive in 39 patients (88.6%), whereas the SVC was positive in 41 patients (93.1%), although the difference was not statistically significant (P = 0.39). It is concluded that the inoculation of all of the leukocytes extracted from blood samples in the SVC seems to produce a slight increase in the sensitivity of the cell culture and that the SVC becomes positive before the antigenemia for the detection of CMV in peripheral blood, especially in the first blood sample.

An increase in the number of polymorphonuclear leukocytes inoculated on shell-vial culture increases the sensitivity of this assay in the detection of cytomegalovirus in the blood of immunocompromised patients.

A prospective study was conducted comparing the sensitivity of the pp65 antigenemia assay (AGA) to that of the shell-vial culture (SVC) inoculated with increasing quantities of polymorphonuclear leukocytes (PMNLs) in the detection of cytomegalovirus (CMV) in peripheral blood. From the cellular suspension, three SVCs were inoculated with 200,000, 400,000, and 800,000 PMNLs, respectively. Of the 201 patients studied, 67 (31.9%) had positive results in one of the two analytic tests (AGA or SVC). In this group, 13 (19.4%) presented a negative AGA assay; 13 (19.4%) an AGA of 1; 13 (19.4%) an AGA of between 2 and 5; and 28 (41.8%) an AGA with a value > 6 PMNL-positive x 100,000 PMNLs. The SVC inoculated with 200,000 PMNLs detected the presence of CMV in 42 cases (62.6%); 55 (82%) with 400,000; and 64 (95.5%) with 800,000. Statistically significant differences were observed between the isolation capacities of the SVC inoculated with 200,000 and 400,000, and the SVC inoculated with 800,000 PMNLs (p = 0.0001). In the comparison of the overall sensitivity of the AGA with that of the SVC with 200,000, the AGA was found to be significantly more sensitive (p = 0.0052). When comparing with the SVC with 400,000 PMNLs, the two techniques were found to be equally sensitive; and in the comparison with the SVC with 800,000, the culture displayed a greater detection sensitivity (p = 0.0023). According to these results, it seems evident that the increase in the absolute number of PMNLs inoculated in the SVC leads to a significant increase in the sensitivity of the SVC in the detection of low-level viremia by CMV.

[Isolation of cytomegalovirus from peripheral blood by shell-vial culture. Technical considerations]. / Aislamiento de citomegalovirus a partir de sangre periférica mediante cultivo en shell-vial. Consideraciones técnicas.

BACKGROUND: We performed an study of the possible benefits derived from the use of two vials, incubated for 24 and 72 h respectively, in the isolation of CMV from the leukocyte polymorphonuclear (LPMN) of peripheral blood (viremia) in immunosuppressed patients, using the shell-vial culture technique. MATERIAL AND METHODS: The blood samples with EDTA were fractionated by sedimentation with saline dextran. The LPMN-rich upper layer was used for the isolation of CMV in the shell-vial cell culture (MRC-5 cell line) and to prepare the antigenemia pp65 assay. The cultures were stained at 18-24 h (first vial) and 72 h (second vial) with an indirect immunofluorescence assay with a monoclonal antibody against p72 CMV antigen. RESULTS: We studied 878 blood samples of which 247 (28.1%) were positive for CMV. Of the positive samples, 211 (85.4%) were detected in the first vial, and 220 (89%) in the second vial. Neither of the two vials detected all positive samples. An overall toxicity of 3%, in the positive samples, and 8.5% in positive samples was detected, respectively. Of the 28 toxic samples, 25.9% were detected in the first vial and 85.1% in the second (p < 0.001). The use of the second vial increased the number of positive samples in 14.5% (36 blood samples). CONCLUSIONS: In view of the results obtained in this study it would seem that, in order to obtain the maximum diagnostic yield in the isolation of CMV from LPMN of peripheral blood in immunosuppressed patients by means of the shell-vial culture, it is necessary to inoculate all the LPMN extracted into two vials. The lengthening of the incubation period up to 72 h for one of the vials leads to an increased in the isolation of CMV of 14.5% with a low overall toxicity.

Comparison of Madin-Darby canine kidney cells (MDCK) with a green monkey continuous cell line (Vero) and human lung embryonated cells (MRC-5) in the isolation of influenza A virus from nasopharyngeal aspirates by shell vial culture.

We report a comparative study of the MDCK, Vero, and MRC-5 cell lines in the isolation of the influenza A (IA) virus. We studied 746 samples in which 63 IA viruses were isolated. The MDCK line displayed 100% sensitivity, the Vero line displayed 71.4%, and the MRC-5 displayed 57.1%. The MDCK line showed a statistically significant difference with respect to the Vero line (P = 0.001) and the MRC-5 line (P = 0.001). The quantitative sensitivity analysis showed the MDCK line to be superior to the other lines. It seems that the MDCK line is still one of the most recommendable for the isolation of the IA virus from respiratory samples.

Evaluation of a direct immunofluorescence cytospin assay for the detection of herpes simplex virus in clinical samples.

A comparison between a direct immunofluorescence assay (DFA) and the shell-vial culture (SVC) was conducted to evaluate their efficacies according to the quality and origin of the sample and the type of herpes simplex (HSV) responsible for the infection. The SVC detected all 58 HSV-infected samples, while the DFA detected only 49 (84.5%) positive samples. The DFA detected HSV type 1 in 22 of 89 samples (24.7%) and HSV type 2 in 27 of 96 samples (28.1%). Compared with the SVC, the DFA had a sensitivity of 75.8% for HSV type 1 and 93.1% for HSV type 2. The sensitivity of the DFA depends on the quality of the sample. Thus, while DFA is recommendable as a screening method, the SVC remains the method of choice for obtaining the maximum diagnostic yield from the sample.

[Presence of virulence factors and pathovars in strains of Campylobacter jejuni isolated from patients with diarrhea and healthy children]. / Presencia de factores de virulencia y patovariedades en cepas de Campylobacter jejuni aisladas en pacientes con diarrea y niños sanos.

BACKGROUND: To study the prevalence of virulence factors (adhesion, invasion, cytotoxicity and hemolytic activity) and to establish the presence of pathovars (virulence phenotype) in C. jejuni strains isolated in pediatric patients with inflammatory and secretory diarrhea and asymptomatic carriers. METHODS: We analyzed 95 strains of 48 patients with inflammatory diarrhea (blood and mucus in feces), 30 patients with secretory diarrhea (watery) and 17 strains isolated in asymptomatic children (control group). The study of adherence capacity, invasion and cytotoxicity was made in the Hep-2 cell line, and the analysis of hemolytic activity in blood agar plates with a 5% sheep's blood. The pathovars were defined by the cellular adhesion (phenotypes A and a) and the cytotoxicity (phenotypes E and e). RESULTS: 29.1% of inflammatory strains presented adherence capacity, 66.6% were invasive, 64.5% cytotoxic and 52.1% hemolytic. In the secretory strains the values were 70, 20, 10 and 6.6% respectively; in the control group the 11.7% presented adherence capacity and 5.8% were invasive. We obtained difference statistically significative for the secretory strains in the adherence capacity, and in inflammatory strains in the adherence capacity, cytotoxicity and hemolysis. The phenotype Ae predominate in the secretory strains, and the phenotype ae in the strains belonging to the control group. No pathovar predominates in the inflammatory strains. CONCLUSIONS: The analysis of virulence markers permit us to establish the pathogenic behaviour of the C. jejuni strains isolated in patients with diarrhea. The study of the adherence capacity and cytotoxicity (pathovars) would be used as a virulence markers and to predict the inflammatory or secretory nature of the diarrhea caused by C. jejuni strains.

In vitro activity of amphotericin B, flucytosine and fluconazole against yeasts causing bloodstream infections.

The in vitro activity of amphotericin B, flucytosine and fluconazole against 95 yeasts causing fungemia in a single institution over the last eight years was determined by a broth macromethod recommended by the National Committee for Clinical Laboratory Standards. All strains were inhibited by amphotericin B concentrations of < or = 1 microgram/ml. With flucytosine in most species the MIC50 was between 0.12 and 0.25 microgram/ml and the MIC90 was between 0.25 and 1 microgram/ml. One exception with flucytosine was Candida krusei, with an MIC50 and MIC90 of 16 micrograms/ml and 32 micrograms/ml, respectively. Overall, 12% of the isolates needed at least 8 micrograms/ml of fluconazole to be inhibited. Fluconazole was very active against Candida albicans, Candida tropicalis and Cryptococcus neoformans, with MIC50 ranging from 0.12 to 0.5 microgram/ml and MIC90 of 1 microgram/ml, and somewhat less active against Candida parapsilosis (MIC50 of 1 microgram/ml and MIC90 of 4 micrograms/ml). Fluconazole exhibited poor in vitro activity against Candida krusei (MIC50 and MIC90 of 64 micrograms/ml) and Torulopsis glabrata (MIC50 of 4 micrograms/ml and MIC90 of 16 micrograms/ml). High MICs of fluconazole were found for four strains of Candida albicans, one with an MIC of 4 micrograms/ml and three (5.7%) with MICs of > or = 16 micrograms/ml. Previous exposure to fluconazole could be demonstrated in two of these strains. Further work must be done in order to determine appropriate breakpoints of antifungal agents, to assess the clinical relevance of azole resistance in yeasts causing bloodstream infections and to identify risk factors for infections with azole-resistant yeasts.

Evolution of susceptibilities of Campylobacter spp. to quinolones and macrolides.

Erythromycin, new macrolides, and quinolones are alternatives for the treatment of Campylobacter infections. Concerns related to the emergence of resistance to both groups of drugs have been raised. We studied the evolution of antimicrobial susceptibilities of 275 clinical isolates of microorganisms of the genus Campylobacter isolated in our institution during a 5-year period (1988 to 1992). The microorganisms studied were C. jejuni (n = 230), C. coli (n = 42), and C. fetus (n = 3). The overall resistance rates (determined by the agar dilution method and the recommendations of the National Committee for Clinical Laboratory Standards) were as follows: erythromycin, 2.3%; clarithromycin, 2.3%; azithromycin, 1.9%; ciprofloxacin, 28.5%; norfloxacin, 31%; ofloxacin, 26.3%; and nalidixic acid, 36.8%. The evolution of resistance (percent resistance in 1988 versus percent resistance in 1992) was as follows: erythromycin, 2.6 versus 3.1; clarithromycin, 2.6 versus 3.1; azithromycin, 2.6 versus 3.1; ciprofloxacin, 0 versus 49.5; norfloxacin, 2.6 versus 55.5; ofloxacin, 0 versus 45.6; nalidixic acid, 2.6 versus 56.8. Our data show stable macrolide activity against Campylobacter spp. and the rapid development of quinolone resistance over the last 5 years.

Treatment of deep mycoses with liposomal amphotericin B.

Amphotericin B is the mainstay of therapy of many deep mycoses, but its use is seriously hampered by dose-limiting nephrotoxicity. In this study a liposomal formulation of amphotericin B was administered to ten patients with proven deep mycoses: invasive aspergillosis (n = 4), deep candidiasis (n = 4) and zygomycosis (n = 2). The mean daily dosage of liposomal amphotericin B was 3.0 mg/kg (range 2.5 to 4 mg/kg), the mean total dosage of liposomal amphotericin B 2,781 mg (range 87 to 5,220 mg) and the mean duration of treatment 17 days (range 3 to 33 days). Treatment with liposomal amphotericin B was associated with little nephrotoxicity and an overall survival rate of 50%. The median increase of serum creatinine from baseline levels was 0.38 mg/dl (-1.2 to 2.6 mg/dl).

Aeromonas peritonitis.

Five new cases of peritonitis caused by Aeromonas species are reported, and 29 others described in the literature are reviewed. Males predominated (71%), and the mean age was 56.9 years. Acquisition was nosocomial in 20% of the cases. All patients except one (3%) had significant underlying diseases; 73% had chronic hepatic disease, 15% had chronic renal failure (treated with chronic ambulatory peritoneal dialysis [CAPD]), and 9% had an intestinal perforation. Symptoms were similar to those of peritonitis caused by other pathogens, with the exception of diarrhea, which occurred in 25% of cases. Blood cultures were positive in 74% of the cases. The species isolated were Aeromonas hydrophila (27), Aeromonas sobria (5), and Aeromonas caviae (2). The overall case-fatality rate was 57%. Three strains were resistant to cotrimoxazole. Aeromonas species should be taken into account as a cause of peritonitis in patients with cirrhosis or who are undergoing CAPD.

[Evaluation of 2 fast methods for the detection of rotaviruses in fecal samples]. / Evaluación de 2 métodos rápidos para la detecciíon de rotavirus en muestras fecales.

BACKGROUND: Rotavirus is one of the major causes of acute gastroenteritis in childhood. Several rapid methods for rotavirus detection are currently available, although their efficacy have a great variability. We performed a prospective study to evaluate two rapid tests for detection of rotavirus antigens in faeces. METHODS: Using electron microscopy as the reference method, we compared two techniques for diagnosis of rotavirus infection: a monoclonal ELISA (Rotaclone, Cambridge Bioscencer, Worcester) and a policlonal latex agglutination (Diarlex, Orion Diagnostics, Finland). We tested, in parallel, 192 stools samples received in our laboratory to investigate rotavirus. RESULTS: ELISA showed significantly better sensitivity than latex (100 vs 66.6%), while both methods had similar specificity (98.6%). CONCLUSIONS: Of the tested methods, ELISA was superior due to the high sensibility and specificity, good reproductibility and easy interpretation. It also permits the simultaneous performance of several tests.