Ultrasensitive digital quantification of cytokines and bacteria predicts septic shock outcomes.

Quantification of pathogen and host biomarkers is essential for the diagnosis, monitoring, and treatment of infectious diseases. Here, we demonstrate sensitive and rapid quantification of bacterial load and cytokines from human biological samples to generate actionable hypotheses. Our digital assay measures IL-6 and TNF-α proteins, gram-negative (GN) and gram-positive (GP) bacterial DNA, and the antibiotic-resistance gene blaTEM with femtomolar sensitivity. We use our method to characterize bronchoalveolar lavage fluid from patients with asthma, and find elevated GN bacteria and IL-6 levels compared to healthy subjects. We then analyze plasma from patients with septic shock and find that increasing levels of IL-6 and blaTEM are associated with mortality, while decreasing IL-6 levels are associated with recovery. Surprisingly, lower GN bacteria levels are associated with higher probability of death. Applying decision-tree analysis to our measurements, we are able to predict mortality and rate of recovery from septic shock with over 90% accuracy.

A preliminary study on the genetic profile of cag pathogenicity-island and other virulent gene loci of Helicobacter pylori strains from Turkey.

Helicobacter pylori genetic diversity affects the function and antigenicity of virulence factors associated with the disease outcome. Gene profile was done to identify the distribution of gene loci within and outside the cag pathogenicity-island (PAI). H. pylori strains from 35 patients [21 gastritis, 14 peptic ulcer diseases (PUD)] were analyzed using PCR. The profile of the cag PAI was evaluated using primers spanning the 3' end, cagA, promoter region of the cagA, cagE, cagT, 5' end (LEC), extreme right end, plasticity region open reading frames (ORFs), oipA (Hp0638) and vacA alleles. We found few intact cag PAI in the strains examined. Deletions were found in LEC1 (9.5% versus 14.3%), LEC2 (4.8% versus 14.3%), cagT (33.3% versus 28.6%), cagE (28.6% versus 28.6%) and the promoter region of the cagA (19.0% versus 42.9%) of gastritis and PUD strains, respectively. The cagA gene was detectable in 57.1% of gastritis and 92.9% of PUD-associated strains. The cagRJ region also showed deletions for many of its genes. The oipA (Hp0638) gene was detected in 80.9% of gastritis and in 92.9% of PUD strains. The plasticity region ORFs JHP912 and JHP931 were predominant in PUD strains. The vacA-s1a-m1a genotype was predominant in PUD, while s2m2 in gastritis strains. This comprehensive analysis showed deletions in several genes within and outside the cag PAI. However, cagA, oipA, JHP912, JHP931 and vacA-s1a-m1a were more predominant in PUD strains than gastritis-associated strains, suggesting the importance of genetic diversity on the disease progression and clinical outcome.

H pylori infection and other risk factors associated with peptic ulcers in Turkish patients: a retrospective study.

AIM: To identify and evaluate the relative impact of H pylori infection and other risk factors on the occurrence of gastric ulcer (GU), duodenal ulcer (DU) and gastritis in Turkish patients. METHODS: A total of 4471 patients (48.3% female) out of 4863 attended the Samatya hospital in Istanbul (June 1999-October 2003) were included. The records of H pylori status (CLO-test), endoscopic findings of GU, DU and gastritis, personal habits (smoking, alcohol intake) and medication [non-steroidal anti-inflammatory drugs (NSAIDs), aspirin intake] were analyzed using multi-way frequency analysis. RESULTS: We have found that GU in the presence of H pylori had significant association with aspirin (P=0.0001), alcohol (P=0.0090) and NSAIDs (P=0.0372). DU on the other hand had significant association with aspirin/smoking/NSAIDs (P=0.0259), aspirin/alcohol (P=0.0002) and aspirin/smoking (P=0.0233), also in the presence of H pylori. In the absence of H pylori GU had significant association with alcohol/NSAIDs (P=0.0431), and NSAIDs (P=0.0436). While DU in the absence of H pylori had significant association with smoking/alcohol/ NSAIDs (P=0.0013), aspirin/NSAIDs (P=0.0334), aspirin/alcohol (P=0.0360). CONCLUSION: In the presence of H pylori, aspirin, alcohol and NSAIDs intake act as an independent risk factors that had an augmenting impact on the occurrence of GU and only together on the occurrence of DU in Turkish patients.

Construction of novel electrochemical immunosensor for detection of prostate specific antigen using ferrocene-PAMAM dendrimers.

In this study, an immunosensor was designed to utilize for the detection of prostate specific antigen (PSA) based on three different generations (G1, G2 and G3) of ferrocene (Fc) cored polyamidiamine dendrimers (Fc-PAMAM) gold (Au) electrode. The self-assembled monolayer principle (SAM) was used to fabricate the sensitive, selective and disposable immunosensor electrodes. In electrode fabrication cysteamine (Cys) was the first agent covalently linked on the Au electrode surface. Immobilized redox center (ferrocene) cored PAMAM dendrimers served as a layer for the further binding of biological components. The monoclonal antibody of PSA (anti-PSA) was covalently immobilized on dendrimers which were attached onto the modified Au surface (Au/Cys/Fc-PAMAMs/anti-PSA). PSA levels were quantitatively analyzed by using electrochemical differential pulse voltammetry (DPV) whose lowest detection limit was calculated as 0.001ngmL(-1). The Au/Cys/FcPAMAM/anti-PSA immunosensor showed excellent performance for PSA at the pulse amplitude; 50mV and the scan rate; 10mV/s in a wide linear concentration range of 0.01ng-100ngmL(-1). Analytical performance and specificity assays were carried out using human serum and different proteins.

Enzyme immunoassay and immunoblotting analysis of Helicobacter pylori infection in Turkish asymptomatic subjects.

Approximately half of the world population is infected with Helicobacter pylori, particularly in developing countries. The aims of the study were to detect H. pylori infection in asymptomatic Turkish subjects, correlate the infection with the associated risk factors, and to evaluate the cytotoxin-associated gene (CagA) status and other H. pylori antigens. Three hundred nine asymptomatic subjects (124 female) 1-82 years of age (average: 31 years) were serologically tested by enzyme immunoassay and immunoblotting. The enzyme immunoassay detected IgG anti-H. pylori antibodies in sera of 216 (70%) out of 309 subjects, 132 (61%) male. Infection rates of 42% in subjects <10 years of age, 55% in 10-19 years, 66% in 20-29 years, 78% in 30-39 years, 79% in 40-49 years, 91% in 50-59 years, 100% in 60-69 years, and 80% in those >70 years of age were detected. Subjects >45 years of age had significantly higher antibody responses, odds ratio = 0.16 (95% confidence interval: 0.07-0.37), than those <45 years. H. pylori infection was significantly higher in married subjects, odds ratio = 0.38 (95% confidence interval: 0.20-0.73), and those with low socioeconomic status. No correlation between gender, education, smoking, and nonsteroidal anti-inflammatory drug intake and infection was detected. Immunoblots revealed antibodies to CagA in 58 (83%) of 70 samples tested. H. pylori infection is prevalent in the asymptomatic Turkish subjects. Marital and socioeconomic status was significantly associated with the acquisition of H. pylori. Antibodies to CagA antigen were highly prevalent in these subjects.

Helicobacter pylori anti-CagA antibodies: prevalence in symptomatic and asymptomatic subjects in Turkey.

BACKGROUND: Several reports have shown the prevalence of anti-CagA antibodies to be associated with the development of peptic ulcer diseases, while others have indicated that there is no such association. AIM: To examine the prevalence of antibodies to CagA and other Helicobacter pylori antigens in symptomatic and asymptomatic subjects in Turkey. subjects and METHODS: Sixty-six symptomatic subjects, 16 to 74 years of age, were examined for H pylori by biopsy-based tests and ELISA. One hundred nineteen asymptomatic subjects, 20 to 65 years of age, were also tested serologically for the presence of H pylori. Samples from both groups that were found to be positive for H pylori by ELISA were then tested by immunoblotting. RESULTS: Fifty-four (82%) symptomatic subjects and 76 (64%) asymptomatic subjects were found to be H pylori-positive by ELISA. Samples from 30 symptomatic subjects who were found to be H pylori-positive by ELISA were analyzed by immunoblotting. Antibodies to CagA (116 kDa) antigen were detected in immunoblots of 11 of 14 (79%) with chronic gastritis, 12 of 13 (92%) with duodenal ulcer and three of three (100%) with gastric cancer. Antigens of the following molecular weights were also detected in these 30 subjects: 89 kDa (VacA) in 21 (70%), 37 kDa in 21 (70%), 35 kDa in 19 (63%), 30 kDa in 27 (90%) and 19.5 kDa in 19 (63%). Immunoblots of 40 ELISA-positive asymptomatic subjects showed that 33 (83%) had antibodies to CagA antigen, 26 (65%) to VacA antigen, 30 (75%) to a 37 kDa antigen, 30 (75%) to a 35 kDa antigen, 39 (98%) to a 30 kDa antigen and 36 (90%) to a 19.5 kDa antigen. CONCLUSIONS: Antibodies to CagA antigen were prevalent in both groups, regardless of the presence of gastroduodenal disease.

Rubella immune status of pregnant and non-pregnant women in Istanbul, Turkey.

OBJECTIVE: Rubella immunization rates are not optimal and infections during pregnancy still occur since many countries incorporate no rubella vaccine in their national immunization program. The evaluation of immunity to rubella virus relies on the presence of specific antibodies. This study was undertaken to determine in a cross-sectional survey whether rubella virus circulation in the Istanbul city, induces detectable immunoglobulin G (IgG) antibodies with a protective level, in a random group of pregnant and non-pregnant women. METHODS: One hundred and sixty women of 20-41-years of age (average 24-years) were grouped as follows: 1. Forty-eight married women. Among these were 41 pregnant women (33 delivered normally, 8 aborted). 2. One hundred and twelve single women. Samples were collected during the periods from October 2000 through to March 2001 and from November 2001 through to May 2002. Rubella specific IgG antibodies were detected (by the ELISA test) in all women tested. RESULTS: Quantitative analysis of the IgG levels showed noticeable variability that ranged between 24-143 IU/ml (average 94). One hundred and forty-five (91%) out of 160 women had rubella IgG levels of above 50 IU/ml with a range of 54-143 IU/ml (average 92) while 15 (9%) had a level between 24-46 IU/ml (average 38). Rubella IgG-avidity test revealed that 116 (73%) of women had high IgG avidity, 22 (14%) had intermediate avidity and 20 (13%) showed low avidity. Two women who were IgM positive, each had either high or intermediate IgG avidity. CONCLUSION: All women tested were seropositive for rubella specific IgG antibodies suggestive of natural virus circulation within the community. Although the majority appeared to possess protective level of such antibodies, screening for protective immunity appears always to be a necessity for future protection against reinfection.

Effect of heat processing on DNA quantification of meat species.

In this study, real-time polymerase chain reaction (PCR) was used for identifying the effects of different temperatures and times of heat treatment on the DNA of meat products. For this purpose, beef, pork, and chicken were baked at 200 °C for 10, 20, 30, 40, 50 min, and for 30 min at 30, 60, 90, 120, 150, 180, 210 °C and also cooked by boiling at 99 °C for 10, 30, 60, 90, 120, 150, 180, 210, and 240 min. The DNA was then extracted from all samples after the heat treatment. Further, a region of 374, 290, and 183-bp of mitochondrial DNA of beef, pork, and chicken, respectively, was amplified by real-time PCR. It was found that baking and boiling of the beef, pork, and chicken resulted in decreases in the detectable copy numbers of specific genes, which varied with the heating time and degree. The results indicated that species determination and quantification using real-time PCR are affected by the temperature, duration of the heat treatment, and size of the DNA fragment to be amplified.

Canonical correlation analysis of factors involved in the occurrence of peptic ulcers.

The impact of risk factors on the development of peptic ulcers has been shown to vary among different populations. We sought to establish a correlation between these factors and their involvement in the occurrence of peptic ulcers for which a canonical correlation analysis was applied. We included 7,014 patient records (48.6% women, 18.4% duodenal ulcer [DU], 4.6% gastric ulcer [GU]) of those underwent upper gastroendoscopy for the last 5 years. The variables measured are endoscopic findings (DU, GU, antral gastritis, erosive gastritis, pangastritis, pyloric deformity, bulbar deformity, bleeding, atrophy, Barret esophagus and gastric polyp) and risk factors (age, gender, Helicobacter pylori infection, smoking, alcohol, and nonsteroidal anti-inflammatory drugs [NSAIDs] and aspirin intake). We found that DU had significant positive correlation with bulbar deformity (P=2.6 x 10(-23)), pyloric deformity (P=2.6 x 10(-23)), gender (P=2.6 x 10(-23)), H. pylori (P=1.4 x 10(-15)), bleeding (P=6.9 x 10(-15)), smoking (P=1.4 x 10(-7)), aspirin use (P=1.1 x 10(-4)), alcohol intake (P=7.7 x 10(-4)), and NSAIDs (P=.01). GU had a significantly positive correlation with pyloric deformity (P=1,6 x 10(-15)), age (P=2.6 x 10(-14)), bleeding (P=3.7 x 10(-8)), gender (P=1.3 x 10(-7)), aspirin use (P=1.1 x 10(-6)), bulbar deformity (P=7.4 x 10(-4)), alcohol intake (P=.03), smoking (P=.04), and Barret esophagus (P=.03). The level of significance was much higher in some variables with DU than with GU and the correlations with GU in spite of being highly significant the majority, were small in magnitude. In conclusion, Turkish patients with the following endoscopic findings bulbar deformity and pyloric deformity are high-risk patients for peptic ulcers with the risk of the occurrence of DU being higher than that of GU. Factors such as H. pylori, smoking, alcohol use, and NSAIDs use (listed in a decreasing manner) are risk factors that have significant impact on the occurrence of DU; aspirin has a significant impact on both DU and GU.

A follow-up study on the effect of Helicobacter pylori eradication on the severity of gastric histology.

Helicobacter pylori genetic diversity and geographic distribution affect the severity of gastric histology; while eradication heals gastritis, the improvement of atrophy and intestinal metaplasia (IM) is still controversial. We investigated whether H. pylori infection and genotypes (cagA-vacA) influence the histological changes and whether eradication resolves these changes. Twenty-one patients (11 duodenal ulcer, 2 gastric ulcer, 8 gastritis) received treatment. Biopsies for CLO, PCR, histology, and culture were collected before and at 1 and 12 months after treatment, and serum samples at 0, 1, 2, 6, and 12 months. H. pylori eradication was achieved in 71% of the patients. Histological scores for H. pylori densities were significantly higher in the antrum and incisura angularis. Scores for mononuclear cell and neutrophil infiltration were significantly higher in regions with a high H. pylori density and improved progressively after eradication. Eight patients with atrophy including five with IM showed no significant changes 12 months after eradication. The cagA gene, detected in 13 (62%), the vacA-sla gene, in 20 (95%), and the vacA-m1 gene, in 12 (57%) of 21 patients were significantly associated with duodenal ulcer. A gradual decline in antibody titer reached an average of 67% 12 months after eradication. H. pylori infection and the associated genotypes (cagA of Western type) affect the severity of the gastric histology (mild forms of atrophy and IM) and the disease outcome. Eradication of H. pylori resulted in healing of gastritis, but with no significant improvement in atrophy or IM.