Preclinical Assessment of a Fully Automated Multiplex PCR Panel for Detection of Central Nervous System Pathogens.

We evaluated a multiplexed PCR panel for the detection of 16 bacterial, viral, and fungal pathogens in cerebrospinal fluid. Panel results were compared to routine testing, and discrepancies were resolved by additional nucleic acid amplification tests or sequencing. Overall, the positive and negative agreements across methods were 92.9% and 91.9%, respectively.

Antigens from Leishmania amastigotes inducing clinical remission of psoriatic arthritis.

A first generation vaccine (AS100-1) was manufactured with protein from four cultured Leishmania species, which proved to be effective in the treatment of psoriasis. A single blind trial on 3,132 psoriasis patients revealed 508 (16.2%) subjects with psoriatic arthritis (PsA) that received AS100-1 antigens. The study group was distributed according to percent psoriasis area and severity index (PASI) reduction from PASI 10 to PASI 100. All groups decreased in arthritis score (AS), tender joints counts and nail changes after treatment; the highest decreased in the PASI 100 group. Relapses of psoriasis and PsA had PASI and AS lower than initial values before treatment. Clinical remissions were at lower doses and less time, after the second course of treatment. Peripheral blood mononuclear cells (PBMC) lymphocyte subsets (LS) varied with PASI range (1-10, 11-20 and 21-72). Pre-treatment, absolute values of gated LS: CD4+, CD8+HLA-, CD8+HLA+, CD8+CD3-, CD8+CD3+ decreased in PBMC as PASI increased, suggesting migration from the blood to the skin. In contrary to the previous finding, the following LS: CD8+CD4-, CD3+CD8-, HLA+CD8-, CD19, CD8+CD4+ and membrane surface immunoglobulin IgA+, IgD+, IgM+, IgE+, and IgG+ increased in PBMC as PASI increased suggesting activation and proliferation by unknown antigens creating a homeostatic cycle between skin/joints and peripheral blood. After nine doses of AS100-1, the following LS: CD8+CD3+, CD8+HLA+, CD3+CD8-, CD4+CD8-, CD8+HLA-, HLA+CD8-, CD8+CD3-, CD19+, CD8+CD4-, CD8+CD4+, IgA+, IgD+, IgM+, IgE+, and IgG+ decreased significantly as compared with values before treatment. The LS decreased stops the vicious cycle between skin/joints and blood explaining clinical remission of lesions.

Antigens from Leishmania amastigotes induced clinical remission of psoriasis.

While injecting volunteers in Venezuela with a vaccine for prevention of leishmaniasis, we observed 100% clinical remission of a psoriatic lesion in one subject. Subsequently, the vaccine (AS100) was evaluated in psoriatic patients with an open label, single center study. The study was conducted in 2,770 subjects and included plaque (79%), guttate (10%), plaque and guttate (10%), palm/plantar, erythrodermia, inverse, plaque and arthritis and nail psoriasis. Baseline PASI compared with post-treatment values were: PASI 100, 23%; PASI 75, 45%; PASI 50, 13%; PASI 10, 9% and

Comparison of fasting and postprandial plasma lipoproteins in subjects with and without coronary heart disease.

Plasma lipoprotein levels, including remnant-like particle (RLP) cholesterol and RLP triglycerides, were assessed in fasting (12 hours) and postprandial (PP) (4 hours after a fat-rich meal) states in 88 patients with coronary heart disease (CHD) and 88 controls. All lipoproteins were assessed by direct methods. We hypothesized that patients with CHD would have greater percent increases in their triglyceride levels, RLP cholesterol, and RLP triglycerides, in response to a fat-rich meal. In the fasting state, triglycerides, RLP cholesterol, RLP triglycerides, and low-density lipoprotein (LDL) cholesterol levels were all significantly higher in cases versus controls by 51%, 35%, 39%, and 40%, respectively. These levels were 57%, 37%, 64%, and 37% higher in the PP state, respectively. Mean high-density lipoprotein (HDL) cholesterol values were 27% lower in cases in both the fasting and PP states. After eating, triglycerides, RLP cholesterol, and RLP triglycerides increased 64%, 71%, and 290% in controls, respectively, whereas in cases these levels increased by 71%, 94%, and 340%, respectively (all p <0.0001). Percent increases in the PP state were not significantly different in cases versus controls. Following the fat-rich meal, LDL and HDL cholesterol decreased by 5% and 4% in controls, and by 7% and 6% in patients, with no significant difference in percent changes between groups. Fasting values correlated very highly with PP values for all parameters (all p <0.0001). Our data indicate that although patients with CHD have higher fasting and PP levels of triglycerides, RLP cholesterol, and RLP triglycerides than controls, the response (percent increase) to a fat-rich meal is comparable in both groups. Thus, a feeding challenge is not essential for assessment of these lipoproteins. Moreover, it is not necessary to obtain a fasting sample to assess direct LDL and HDL cholesterol.

Apolipoprotein E genotype and cardiovascular disease in the Framingham Heart Study.

BACKGROUND: Apolipoprotein (apo) E is a constituent of lipoproteins with considerable variation due to cysteine-arginine exchanges. The apo E4 (Arg112-Cys) polymorphism has been associated with dementia and hypercholesterolemia. We investigated the relation of APOE genotype to cardiovascular disease (CVD) in the Framingham Offspring Study. METHODS AND RESULTS: DNA was isolated from 3413 study participants and APOE genotypes were determined utilizing the polymerase chain reaction and restriction isotyping. In the entire group of subjects, 20.7% had apo E4/4 or E3/4 (Group E4); 14.1% had apo E2/2 or E2/3 (Group E2) and 63.9% had the apo E3/3 genotype (Group E3). Subjects with E2/4 (1.3%) were excluded. Period prevalence of CVD between examinations 1 and 5 (1971-1994) (366 events) was related to APOE genotype. Age adjusted period prevalence of CVD in men was 18.6% for Group E4, 18.2% for Group E2 and 12.7% for Group E3 (P=0.004); while in women these rates were 9.9, 4.9, and 6.6%, respectively (P=0.037). After adjustment for non-lipid risk factors the relative odds for CVD in Group E2 men was 1.79 (P=0.0098) and in Group E4 it was 1.63 (P=0.0086) compared with the Group E3; while in Group E4 women it was 1.56 (P=0.054). After adjustment for all CVD risk factors, the relative odds in Group E2 men was 1.94 (P=0.004) and in Group E4 men it was 1.51 (P=0.0262). CONCLUSIONS: The presence of the apo E2 or apo E4 alleles in men is associated with significantly greater CVD risk. This genotypic information may help to identify individuals at increased risk for CVD events.

In vitro activity of meropenem, imipenem, cefepime and ceftazidime against Pseudomonas aeruginosa isolates from cystic fibrosis patients.

We studied 67 Pseudomonas aeruginosa isolates from cystic fibrosis patients, and compared their in vitro susceptibility to two carbapenems (meropenem and imipenem) and two cephalosporins (cefepime and ceftazidime). The carbapenems were more effective in vitro than the cephalosporins: 92.5% of isolates were susceptible to the former and 77.6% to the latter. Essentially no difference was found between meropenem and imipenem. More discrepancies were seen between cefepime and ceftazidime: four of 67 isolates (6.0%) were more susceptible to cefepime than to ceftazidime, while eight (11. 9%) were more susceptible to ceftazidime than to cefepime.

Comparison of sorbitol MacConkey agar and a two-step method which utilizes enzyme-linked immunosorbent assay toxin testing and a chromogenic agar to detect and isolate enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) and specifically serotype O157:H7 are a significant cause of hemorrhagic gastrointestinal disease and the hemolytic uremic syndrome. Methods currently used in clinical microbiology labs, such as sorbitol-MacConkey (SMAC) agar, reliably detect only O157:H7. We have evaluated a two-step method that has the potential to identify and isolate all EHEC serotypes, including serotype O157:H7. This method utilizes a chromogenic selective-differential medium for the isolation of E. coli together with an enzyme-linked immunosorbent assay (ELISA) that detects the Shiga-like toxins Stx1 and Stx2. Both are commercially available and usable in a wide range of clinical microbiology laboratories. Compared to a Vero cell cytotoxic assay, SMAC had sensitivities of 23.5% for the identification of all EHEC serotypes and of 50.0% for the identification of O157:H7 alone. The two-step method had sensitivities of 76.5 and 100%, respectively. The ELISA alone had a sensitivity of 82.4% in the detection of Stx1 and Stx2. The specificity was 100% in all cases. Overall, 14 EHEC isolates were obtained: 8 (58%) O157:H7, 2 (14%) O26, 2 (14%) O111:NM, 1 (7%) O103:H2, and 1 (7%) O121:H19. All but one were isolated during the months of May to September. The two-step method was found to be considerably more expensive than SMAC for both positive and negative samples.

Overview of some biomedical research projects in tropical medicine conducted at the Instituto Venezolano de Investigaciones Cientificas.

The Instituto Venezolano de Investigaciones Cientificas (IVIC) is a government-funded multidisciplinary academic institution dedicated to research, development and technology in many areas of knowledge. Biomedical projects and publications comprise about 40% of the total at IVIC. In this article, we present an overview of some selected research and development projects conducted at IVIC which we believe contain new and important aspects related to malaria, ancylostomiasis, dengue fever, leishmaniasis and tuberculosis. Other projects considered of interest in the general area of tropical medicine are briefly described. This article was prepared as a small contribution to honor and commemorate the centenary of the Instituto Oswaldo Cruz.

Bacillus cereus infections among oncology patients at a children's hospital.

BACKGROUND: Bacillus cereus can cause severe infections in immunocompromised persons. METHODS: We report 3 cases of bacteremia/septicemia (1 fatal) among oncology patients in a children's hospital. Because all cases occurred during a 10-day period, a common source outbreak was suspected. An epidemiologic investigation was performed. Molecular comparison of patient and environmental isolates was performed by using pulsed-field gel electrophoresis. RESULTS: After an extensive investigation, no common hospital source could be found. Pulsed-field gel electrophoresis proved that the isolates were not related. CONCLUSION: Sporadic infections in immunocompromised persons do occur and can be associated with significant morbidity.

Detection of vancomycin-resistant enterococci colonization in a children's hospital.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens in many hospitals. The true prevalence of VRE in pediatric hospitals is not known. METHODS: A surveillance study was performed at a pediatric tertiary care medical center by using vancomycin-containing screening media. RESULTS: Six children (of 112 screened) were found to be colonized with VRE. Colonized patients had a history of receiving broad-spectrum antimicrobial agents. CONCLUSION: In the absence of VRE infections, surveillance studies can help determine the extent of VRE colonization and support infection control measures.

Epidemiologic characterization of American cutaneous leishmaniasis in an endemic region of eastern Venezuela.

The status of American cutaneous leishmaniasis was investigated from 1985 to 1991 to provide an epidemiologic characterization of the disease in Bergantin, a rural community in the northeastern part of Anzoátegui State, Venezuela. The study revealed the presence of the infection during the period analyzed, with an average incidence of 50.2 cases per 10,000 inhabitants and this number has increased 1.5 times during the last two years. Three villages where clinical cases had been recorded were selected for a comparison of their prevalence data. These villages comprise the human population in the high and low altitude limits of Bergantin. Immunologic assessment of the inhabitants used two different antigen preparations to examine responses to parasites associated with the cutaneous and visceral forms of the disease. The leishmanin skin test (LST) was used in a sample of 276 individuals (46.3% of the inhabitants) and resulted in an overall positivity of 16.7%. The percentage of LST positivity varied with age and sex, yet analysis of this response and the prevalence for each village reflected the specific characteristics of these localities. La Montaña, situated at 800 meters above sea level, had the highest prevalence (800 cases per 10,000 inhabitants) and the most positive LST response (21.2%) in comparison with the two other villages situated at a lower altitude (300 meters above sea level).

Evaluation of the BIOMIC video reader system for determining interpretive categories of isolates on the basis of disk diffusion susceptibility results.

The BIOMIC System (Giles Scientific, New York, N.Y.) includes software and a video-assisted plate reader that functions with a personal computer to automate, speed read, and interpret standard antibiotic disk diffusion test plates. The video reader helps standardize endpoints, speeds quantitative measurements by 40 to 90%, and reduces fatigue and transcription and interpretation errors (H. Wei-Fang, Am. Clin. Lab. 13:28-29, 1994). Organisms tested were isolated from patient specimens collected at Primary Children's Medical Center and included rapidly growing gram-positive and gram-negative strains that fulfill the National Committee for Clinical Laboratory Standards guidelines for disk diffusion susceptibility testing. A comparison of the plate reader-determined zones and visually measured zones for 3,339 organism-antimicrobial agent combinations was performed. The results demonstrated 0.1% (4 of 3,339) false-susceptible reads and 0.2% (6 of 3,339) false-resistant reads by the video reader compared with visual reads. Minor discrepancies (4.7% [156 of 3339]), resulting in category interpretation changes of intermediate to resistant or susceptible or changes of resistant or susceptible to intermediate, were also encountered. Of the discrepant results, 80.8% (139 of 172) resulted from a 3-mm or less zone diameter difference between the two different techniques. We conclude that the video-assisted plate reader is a reliable system for determining interpretative categories from zone diameters of standard antibiotic disk diffusion test plates.

Surveillance and detection of erythromycin resistance in Bordetella pertussis isolates recovered from a pediatric population in the Intermountain West region of the United States.

Forty-seven Bordetella pertussis isolates recovered from January 1985 to June 1997 at Primary Children's Medical Center were tested for erythromycin resistance. Agar dilution MICs were determined on Regan-Lowe agar. Forty-six isolates were found to be erythromycin susceptible (all MICs were less than or equal to 0.12 microg/ml). One isolate was found to be erythromycin resistant (MIC, 32 microg/ml). In addition, we compared Etest MIC results and disk diffusion zone diameter measurements, performed on commercially prepared Regan-Lowe agar, to the agar dilution MIC result. Etest MIC and/or disk diffusion testing on commercial Regan-Lowe agar appears to be an adequate method for erythromycin resistance screening of B. pertussis isolates.

Meningitis due to Ochrobactrum anthropi: an emerging nosocomial pathogen. A report of 3 cases.

We describe 3 cases of Ochrobactrum anthropi meningitis following the implantation of pericardial allograft tissue to cover dural defects following craniotomy. Following an extensive epidemiologic investigation, the tissue allograft was found to have been contaminated with this unusual organism during the harvesting and processing of the tissue in the tissue bank. This organism was only susceptible to imipenem, tetracycline, gentamicin, and ciprofloxacin. The clinical presentation of these patients was subacute. Two of the patients developed osteomyelitis of the bone flap; while another developed a relapse of infection along a former ventriculoperitoneal shunt track 6 months after the initial infection. Appropriate clinical outcome was only observed after removal of tissue allograft implants, debridement of devitalized tissue and bone, removal of shunt devices, and prolonged courses of antibiotics. No deaths were observed.

Ochrobactrum anthropi meningitis in pediatric pericardial allograft transplant recipients.

An epidemiologic investigation was done after 3 patients contracted Ochrobactrum anthropi meningitis at one hospital in October 1994. Neurosurgical patients with pericardial tissue implants were at greater risk of infection than other neurosurgical patients (3/14 vs. 0/566; P<.001). Cultures of implants removed from 2 case-patients, an implant at implantation, a nonimplanted pericardial tissue, and an unwrapped but unopened bottle of Hank's balanced salt solution (HBSS) grew O. anthropi. Patient and tissue isolates had identical genotypes; the isolate from the HBSS bottle had a unique genotype. Culture samples from an unopened HBSS bottle and from pericardial tissue grew Pseudomonas stutzeri of the same genotype; however, no P. stutzeri infections were detected. The investigation documented intrinsic P. stutzeri contamination of HBSS. O. anthropi contamination of tissues occurred during processing, possibly due to extrinsic contamination of HBSS. Active surveillance is needed to detect infection in patients receiving transplanted tissues, and rigorous infection control practice are necessary during tissue harvesting and processing to ensure sterility.

Comparison of chagasic and non-chagasic myocardiopathies by ELISA and immunoblotting with antigens of Trypanosoma cruzi and Trypanosoma rangeli.

Trypanosoma cruzi associated myocardiopathy, or Chagas disease, continues to be a serious problem in Venezuela, for which there is neither a vaccine nor a cure. In order to learn more about the humoral immune response to trypanosomal antigens, and to try to identify dominant antigens, we used ELISA and immunoblotting to study the reactivity of sera from patients with chagasic and non-chagasic myocardiopathies, against surface and secreted proteins from T. cruzi and T. rangeli. Both species are found in the same insect vector, but only T. cruzi is thought to be pathogenic in vertebrates. The ELISA results fell into three patterns: (1) high reactivity values with both T. cruzi and T. rangeli surface and secreted proteins; (2) high values to T. cruzi but low values with T. rangeli; and (3) high values to T. rangeli and low values with T. cruzi. This finding that some chagasic sera react more strongly against T. rangeli than against T. cruzi is intriguing, and warrants further investigation. When chagasic sera were tested on Western blots of total extracts of T. cruzi and T. rangeli, the pattern of reactive bands was similar against both parasites, but no two sera showed an identical pattern. Furthermore, there was no correlation between a particular immunoblotting pattern and either the antibody titer, or the severity of the disease. Several T. cruzi and T. rangeli antigens were recognized by sera from healthy controls as well as from patients with other tropical diseases endemic in Venezuela. Overall, our results suggest that the humoral immune response to trypanosomal antigens is complex, and no single antigen may be the determining factor in the pathogenesis of chagasic myocardiopathy.

Rapid diagnostic tests in microbiology in the 1990s.

The selection and management of all diagnostic tools should be guided in the 1990s by carefully informed decisions. Concerns of test sensitivity and specificity must be balanced against cost. Although clinical judgment alone is often prompt and cost-effective, the risk of inaccuracy and serious consequences may be too great to forgo rapid diagnostic assays. The clinical effect and the imperatives in health care of the 1990s will guide the intelligent management and use of advancing rapid assay technology.

Optical immunoassay for streptococcal pharyngitis: evaluation of accuracy with routine and mucoid strains associated with acute rheumatic fever outbreak in the intermountain area of the United States.

The Strep A OIA (BioStar, Inc., Boulder, Colo.) rapid detection system is an intriguing technology that utilizes an immunoassay relying on changes in reflected light to directly detect group A streptococcal antigen from specimens. In this evaluation, 424 routine pediatric throat specimens and 20 simulated oropharyngeal specimens with added mucoid (M type 3, 18) strains were cultured and tested by the Strep A OIA. The respective sensitivities and specificities were as follows: Strep A OIA versus enhanced broth culturing, 84.2 and 95.7%; and streptococcus-SXT agar (BBL Microbiology Systems, Cockeysville, Md.) culturing versus enhanced broth culturing, 82.9 and 98.6%. The Strep A OIA is an 8-min, technologist-friendly, accurate technique with an 89.4% agreement with traditional culturing.

Rapid detection of Streptococcus pyogenes in pediatric patient specimens by DNA probe.

A chemiluminescent DNA probe test (Group A Streptococcus Direct Test; Gen-Probe, Inc., San Diego, Calif.) for rapid, direct detection of cRNA of Streptococcus pyogenes in throat swabs was compared with conventional culture and identification techniques. Throat swabs from 277 patients suspected of having streptococcal pharyngitis were examined. By DNA probe alone, 10 specimens were positive, 51 were positive by both assays, and 8 were positive by culture alone. Thus, DNA probe sensitivity, specificity, and positive and negative predictive values were 86, 95, 84, and 96%, respectively. Including an indeterminate category, sensitivity, specificity, and positive and negative predictive values were 89, 96, 86, and 97%, respectively. After discrepancy testing, these values for the raw data improved to 90, 98, 93, and 97%, respectively. None of the 24 specimens that grew non-S. pyogenes beta-hemolytic streptococci in culture were positive by the DNA probe. Because mucoid S. pyogenes strains are more virulent than nonmucoid strains, 24 isolates were retrospectively tested with the DNA probe to ensure that both types would be detected equally well. Isolates were examined in pure cultures as well as mixed with representative normal oral flora. There was no statistical difference in detection of any of the four groups. Group A Streptococcus Direct Test is a rapid, sensitive, and specific test for S. pyogenes.

A comparison of the molecular biology of trypanosomes and leishmaniae, and its impact on the development of methods for the diagnosis and vaccination of leishmaniasis and Chagas disease.

Our main interest have focused on Chagas disease and Leishmaniasis, working in the areas of: 1--The molecular biology of Trypanosomes and Leishmaniae, and 2--The immunology of Chagas disease, cutaneous leishmaniasis and visceral leishmaniasis. In this article we summarize the work realized in the last 20 years in the Immunobiology Laboratory at the IVIC with special emphasis in the development of a vaccine against leishmaniasis that is being currently used in a field trial in human beings of the endemic area of Guatire, Miranda State, Venezuela.