Rapid and Sensitive Multiplex Detection of Burkholderia pseudomallei-Specific Antibodies in Melioidosis Patients Based on a Protein Microarray Approach.

BACKGROUND: The environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking. METHODS AND PRINCIPAL FINDINGS: In this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response. CONCLUSIONS: Our protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-B. pseudomallei antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.

DNA microarray-based PCR ribotyping of Clostridium difficile.

This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray.

Development and usage of protein microarrays for the quantitative measurement of Panton-Valentine leukocidin.

Staphylococcus aureus is a human pathogen that can harbour several genes encoding exotoxins including leukocidins. A clinically most relevant factor is Panton-Valentine leukocidin (PVL) because of its association with chronic, recurrent or severe skin and soft tissue infections. In this study an antibody array was designed and used to obtain an overview about the in vitro PVL expression levels of 266 clinical isolates of MRSA as well as of MSSA belonging to a wide variety of clonal complexes. For that purpose, a novel precipitation based method was used. Unknown PVL concentrations were determined by mapping the signal intensities for spotted monoclonal antibodies to calibration curves that resulted from experiments with known concentrations of recombinant LukF-PV. In most cases, isolates belonging to one clonal complex (CC) showed similar PVL expressions. However, there were also CCs with widely varying PVL concentrations. First analyses, based on in vitro PVL measurements, showed low PVL concentrations in isolates from severe and fatal conditions that are not associated with PVL, such as sepsis, while isolates from skin and soft tissue infections yielded higher concentrations. Agr-group I and IV isolates generally produced more PVL than isolates from agr-groups II and III. The few isolates harbouring the gene encoding toxic shock syndrome toxin (tst1) were particularly low level PVL producers. However, these issues warrant further studies. The method described herein allows rapid quantification of expressed proteins such as PVL in collections of clinical isolates in order to correlate with clinical or genotypic data with a potential for further parallelisation.

Genome sequencing and molecular characterisation of Staphylococcus aureus ST772-MRSA-V, "Bengal Bay Clone".

BACKGROUND: The PVL-positive ST772-MRSA-V is an emerging community-associated (CA-) MRSA clone that has been named Bengal Bay Clone since most patients have epidemiological connections to the Indian subcontinent. It is found increasingly common in other areas of the world. METHODS: One isolate of ST772-MRSA-V was sequenced using the Illumina Genome Analyzer System. After initial assembling the multiple sequence contigs were analysed using different in-house annotation scripts. Results were compared to microarray hybridisation results of clinical isolates of ST772-MRSA-V, of related strains and to another ST772-MRSA-V genome sequence. RESULTS: According to MLST e-burst analysis, ST772-MRSA-V belongs to Clonal Complex (CC)1, differing from ST1 only in one MLST allele (pta-22). However, there are several additional differences including agr alleles (group II rather than III), capsule type (5 rather than 8), the presence of the egc enterotoxin gene cluster and of the enterotoxin homologue ORF CM14 as well as the absence of the enterotoxin H gene seh. Enterotoxin genes sec and sel are present. ST772-MRSA-V harbours the genes encoding enterotoxin A (sea) and PVL (lukS/F-PV). Both are located on the same prophage. CONCLUSIONS: ST772-MRSA-V may have emerged from the same lineage as globally spread CC1 and CC5 strains. It has acquired a variety of virulence factors, and for a CA-MRSA strain it has an unusually high number of genes associated with antibiotic resistance.

Decreased baroreflex sensitivity in acute schizophrenia.

Decreased vagal activity has been described in acute schizophrenia and might be associated with altered cardiovascular regulation and increased cardiac mortality. The aim of this study was to assess baroreflex sensitivity in the context of psychopathology. Twenty-one acute, psychotic, unmedicated patients with a diagnosis of paranoid schizophrenia were investigated after admission to the hospital. Results were compared with 21 healthy volunteers matched with respect to age and sex. Cardiovascular parameters obtained included measures for heart rate variability, baroreflex sensitivity, as well as cardiac output, left ventricular work index, and total peripheral resistance. All parameters investigated were analyzed using linear and novel nonlinear techniques. Positive and negative symptoms were assessed to estimate the impact of psychopathology on autonomic parameters. Subjects with acute schizophrenia showed reduction of baroreflex sensitivity accompanied by tachycardia and greatly increased left ventricular work index. Nonlinear parameters of baroreflex sensitivity correlated with positive symptoms. For heart rate variability, mainly parameters indicating parasympathetic modulation were decreased. Vascular pathology could be excluded as a confounding factor. These results reflect a dysfunctional cardiovascular regulation in acute schizophrenic patients at rest. The changes are similar to adaptational regulatory processes following stressful mental or physical tasks in healthy subjects. This study suggests that hyperarousal in acute schizophrenia is accompanied by decreased efferent vagal activity, thus increasing the risk for cardiovascular mortality. Future studies are warranted to examine the role of the sympathetic system and possible autonomic differences in hyperarousal induced by anxiety and/or external stressful events.

Heart rate variability, blood pressure variability, and baroreflex sensitivity in overtrained athletes.

OBJECTIVE: To assess the effects of abruptly intensified physical training on cardiovascular control. DESIGN: Retrospective longitudinal study. SETTING: Research laboratory. PARTICIPANTS: Ten healthy athletes (5 men and 5 women) from track and field as well as triathlon. INTERVENTIONS: A 2-week training camp, including daily stepwise increasing cycling tests, running of 40 minutes, and additional cycling of 60 minutes. MAIN OUTCOME MEASUREMENTS: Time and frequency domain parameters of resting heart rate and blood pressure variability (HRV and BPV) and baroreflex sensitivity (BRS), before, during, and after the training camp. RESULTS: We found significantly reduced HRV during the training camp (mean beat-to-beat interval: 1042 [937 to 1194] ms vs. 933 [832 to 1103] ms vs. 1055 [947 to 1183] ms, P < 0.01; root-mean-square of beat-to-beat interval differences: 68 [52 to 95] ms vs. 52 [38 to 71] ms vs. 61 [48 to 78] ms, P < 0.05). Further, BRS was significantly reduced: 25.2 (20.4 to 40.4) ms/mmHg vs. 17.0 (12.9 to 25.7) ms/mmHg vs. 25.7 (18.8 to 29.1) ms/mmHg, P < 0.05. These effects disappeared at a large degree after 3 to 4 days of recovery. CONCLUSION: Abruptly intensified physical training results in an altered autonomic cardiovascular activity towards parasympathetic inhibition and sympathetic activation that can be monitored by means of HRV and BRS analyses and might provide useful markers to avoid the overtraining syndrome.

Heart rate variability and sympathetic skin response in male patients suffering from acute alcohol withdrawal syndrome.

BACKGROUND: Many symptoms of alcohol withdrawal (AW) such as tachycardia or elevated blood pressure might be explained by increased peripheral and central adrenergic activity. In contrast to many neurochemical studies of sympathetic activation during AW, only very few studies investigated autonomic balance using neurophysiological methods. METHODS: We investigated heart rate variability (HRV) and sympathetic skin response (SSR) in male patients suffering from mild AW syndrome (n = 20, no treatment required) and in patients with moderate to severe AW syndrome (n = 20, clomethiazole treatment) in the acute stage. Sympathovagal influence was quantified using measures of time and frequency domain of HRV as well as modern nonlinear parameters (compression entropy). Furthermore, we obtained latencies and amplitudes of SSR to quantify isolated sympathetic influence. Measures were obtained during the climax of withdrawal symptomatology before treatment, 1 day after climax, and shortly before discharge from hospital. Alcohol withdrawal scores were obtained and correlated to autonomic measures. RESULTS: Ambulatory blood pressure and AW scores revealed characteristic withdrawal symptoms in both patient groups. Apart from the nonlinear parameter compression entropy, Hc, measures of HRV revealed no sign of autonomic dysfunction in contrast to the significantly increased heart rates at the time of admission. Latencies and amplitudes of SSR did not indicate any increase of sympathetic activity. A negative correlation was found between Hc and mental withdrawal symptoms. CONCLUSIONS: We show here that classical measures for autonomic nervous system activity such as HRV and SSR are not suitable for describing the autonomic changes seen in acute AW, although a major role for the sympathetic nervous system has been proposed. This might be due to multiple dysregulation of metabolites in AWS or to subtle alcohol-induced damage to neuronal structures, issues that should be addressed in future studies.

Reduced baroreflex sensitivity in acute alcohol withdrawal syndrome and in abstained alcoholics.

Acute alcohol withdrawal is often associated with increased sympathetic activity, and a decreased baroreflex sensitivity (BRS) can be assumed. Parameters of heart rate variability (HRV), blood pressure variability (BPV), BRS as well as cardiac index (CI), left ventricular work index (LVWI) and total peripheral resistance (TPR) were investigated in 20 patients undergoing acute alcohol withdrawal and matched controls. Measures were obtained during the peak of withdrawal symptomatology prior to treatment as well as 2 and 24h under continuous clomethiazole treatment. Alcohol withdrawal scores were obtained and correlated with autonomic measures. In addition, parameters were assessed in 15 subjects who abstained from alcohol after long-term intake. We found a severe down-regulation of BRS during acute alcohol withdrawal and to a milder extent in abstained alcoholics. Furthermore, HRV and BPV did not unequivocally reveal signs of elevated sympathetic activity. Non-linear parameters of HRV and parameters of BRS correlated with the severity of AWS. The distinct decrease of BRS in AWS and in long-term abstained subjects described here is of importance since similar alterations have been identified as independent prognostic factors for cardiac mortality in other diseases.

Altered autonomic cardiac control predicts restenosis after percutaneous coronary intervention.

BACKGROUND: Early and late restenosis in up to 30% remains a major problem for long-term success after percutaneous coronary intervention (PCI). Compared to bare metal stents, the use of drug-eluting stents reduces restenosis below 10%, but implant coasts have to be considered. In restenosis noninvasive testing lacks diagnostic power. We applied a new approach to identify patients with a high risk for restenosis after PCI by combining heart rate (HR) and blood pressure variability (BPV) analyses. METHODS: In 52 patients with clinical suspicion of restenosis and history of PCI, we investigated patterns of cardiovagal autonomic regulation prior to cardiac catheterization. The patients were separated in (i) patients with restenosis (CAD+R) and (ii) patients without restenosis (CAD-R), where restenosis is defined as a stenosis greater than 75% of luminal diameter in at least one main vessel. The following parameters/methods were evaluated: Canadian Cardiovascular Society grade (CCS-grade), vessel disease score (CAD-level), left ventricular ejection fraction (LVEF), heart rate variability (HRV), BPV, baroreflex sensitivity (BRS), as well as HR turbulence and blood pressure (BP) potentiation caused by premature ventricular complexes. RESULTS: Whereas age, LVEF, CAD-level, CCS-grade, and mean BP did not differ between CAD+R and CAD-R, significant differences were found in (i) BPV: diastolic LF/P, systolic, and diastolic UVLF, (ii) in BRS: slope of tachycardic sequences, and (iii) in extrasystolic parameters: heart rate turbulence onset (HRTO) and potentiation of systolic BP (SBPP). Standard HRV parameters did not show significant differences between the groups. Using the two parameters diastolic LF/P (threshold >0.2) and HRTO (threshold >0) restenosis were predicted in 83.4%. CONCLUSIONS: These results demonstrate that indicators of sympathetic activation or vagal depression identify restenosis in patients after PCI, thus opening a perspective for a new noninvasive monitoring.

Autonomic cardiovascular control in pregnancies with abnormal uterine perfusion.

BACKGROUND: Abnormal uterine perfusion is associated with the development of hypertensive pregnancy disorders. However, its impact on maternal autonomic cardiovascular control is poorly understood. The aim of our study was to investigate the adaptation of autonomic control during pregnancy with abnormal uterine perfusion with normal and pathologic outcome in comparison to normal pregnancies. based on blood pressure variability (BPV), baroreflex sensitivity (BRS), and heart rate variability (HRV) analyses. METHODS: A total of 32 healthy pregnant women (CON, age 28 years, range 24 to 31 years); 16 women with abnormal uterine perfusion and normal outcome (AP-NO, age 29 years, range 28 to 33 years); and 19 women with abnormal uterine perfusion and pathologic outcome (AP-PO, age 26 years, range 25 to 30 years), were recruited for this longitudinal study. Beginning in the 20th week of pregnancy, the women were monitored every fourth week until delivery. For the analysis of BPV, BRS, and HRV, high-resolution ECG, and noninvasive continuous blood pressure (BP) recordings were taken simultaneously for 30 minutes. RESULTS: CON showed pregnancy-induced adaptation of cardiovascular control; in the course of gestation BPV was increased while parameters of HRV and BRS were reduced. On the contrary, no changes during the second half of pregnancy could be observed in pregnancies with abnormal perfusion. Variability parameters were significantly altered in women with abnormal perfusion compared with CON, whereas these changes were more pronounced in AP-PO compared with AP-NO. CONCLUSIONS: Abnormal uterine perfusion, independently of the pregnancy outcome, has a significant impact on maternal cardiovascular control. Measures of BPV, BRS and HRV might be used for improved risk stratification.

Short- and long-term joint symbolic dynamics of heart rate and blood pressure in dilated cardiomyopathy.

Autonomic cardiovascular control involves complex interactions of heart rate and blood pressure. In patients with dilated cardiomyopathy (DCM), this control is impaired and parameters for its quantification might be of prognostic importance. In this paper, we introduce methods based on joint symbolic dynamics (JSD) for the enhanced analysis of heart rate and blood pressure interactions. To assess the coarse-grained dynamics beat-to-beat changes of heart rate and blood pressure are encoded in symbol strings. Subsequently, the distribution properties of short symbol sequences (words) as well as the scaling properties of the whole symbol string are assessed. The comparison of joint symbolic heart rate and blood pressure dynamics in DCM (n = 75) with those in healthy controls (n = 75) showed significant changes. Both, the distribution of words and the scaling properties indicate a loss in heart rate dynamics associated with blood pressure regulation in DCM. In conclusion, the analyses of short- and long-term JSDs provide insights into complex physiological heart rate and blood pressure interactions and furthermore reveal patho-physiological cardiovascular control in DCM.

Smelling renal dysfunction via electronic nose.

The human body odor plays an important role in social communication in various situations, like the olfactory identification of partners and relatives as well as in parents-child interactions. In patients with renal dysfunction the compound of sweat and volatile gases is changed because of the limited ability for removing metabolic products from the blood. The regulation of electrolyte composition and acid-base balance are also altered so that the body odor of these patients may be significantly influenced by these disorders. We show the ability of an electronic nose to detect changes in the human body odor in consequence of renal dysfunction by reducing multivariate sensor signals with principal component analysis to its first and second principal odor component (POC). All healthy subjects could clearly be distinguished from patients with renal failure using quadratic discriminant analysis, whereas a correct classification of 95.2% (98.4% using 1st-3rd POC) of patients between end stage renal failure and chronic renal failure was found. This methodology of analyzing human body odor may also provide new approaches for investigating symptoms of renal failure and for diagnosing other diseases of internal or cutaneous origin.

Alternans of blood pressure and heart rate in dilated cardiomyopathy.

Impaired myocardial performance is known to be associated with electrical and mechanical beat-to-beat alternans phenomena. The alternans in blood pressure and heart rate and their interdependency in idiopathic dilated cardiomyopathy (IDC) were studied. The arterial blood pressure and the electrocardiograph (ECG) were continuously recorded in 22 patients suffering from IDC (age 49 +/- 13 years, ejection fraction 0.33 +/- 0.13, left ventricular diameter of 67 +/- 8 mm) and in 21 healthy controls (age 52 +/- 15 years). The beat-to-beat variations of the interbeat intervals (IBI) and of the blood pressure amplitudes (AMP) were measured. An alternans beat was defined as a beat preceded and followed by beats that had higher or lower values in the respective modality. The percentages of singular and repetitive alternans patterns, and the interdependency of the alternans patterns in AMP and IBI were assessed. The study found significantly more singular and repetitive alternans patterns in the IDC group compared to the control group both in the analysis of AMP and IBI (singular alternans in IBI: 55 +/- 11 vs 47 +/- 7%, P < 0.01; singular alternans in AMP: 61 +/- 15 vs 45 +/- 6%, P < 0.01; triple alternans in IBI: 29 +/- 18 vs 16 +/- 9%, P < 0.01; triple alternans in AMP: 34 +/- 24 vs 12 +/- 7%, P < 0.01). The amplitudes of the AMP alternans patterns were higher in IDC compared to controls (9 +/- 7 vs 4 +/- 2% of AMP, P = 0.01) whereas they did not differ in IBI. The correlation analysis revealed a significant interdependency of the alternans pattern in IBI and AMP in 18 of 22 IDC patients and in 12 of 21 controls (r = 0.50 +/- 0.21 [IDC]; r = 0.26 +/- 0.05 [controls]). The slope of the linear regression (delta AMP vs delta IBI) was steeper in the IDC group compared to the control group (62 +/- 50 vs 20 +/- 22 mmHg/s, P < 0.01). The percentages of alternans patterns appearing in IBI and AMP were positively correlated to the left ventricular diameter (r = 0.70 in the IBI, and r = 0.30 in the AMP). The blood pressure amplitude and the heart rate did not differ between the two groups. Patients suffering from IDC have a higher prevalence, stability, amplitude, and interdependency of alternans patterns in IBI and AMP compared to the control group. The amount of alternans patterns indicates the stage of disease. The alternans analysis may have impact on the functional assessment of patients suffering from heart failure.

Postextrasystolic regulation patterns of blood pressure and heart rate in patients with idiopathic dilated cardiomyopathy.

Assessment of fluctuations in heart rate (HR) following a premature ventricular complex (PVC) is valuable for identifying patients at high risk of sudden cardiac death. We hypothesised that postextrasystolic potentiation is the main determinant of the regulation patterns of blood pressure (BP) and HR following a PVC. Twelve patients with idiopathic dilated cardiomyopathy (IDC) and 13 control subjects with single PVCs (comparable coupling intervals) were investigated. Non-invasive finger arterial BP and ECGs were analysed. Regulation patterns following a single PVC were quantified using the indices postextrasystolic amplitude potentiation (PEAP) and maximum turbulence slope of five consecutive mean BP values (MBP-TS), and compared with the HR turbulence parameters turbulence slope (HR-TS) and turbulence onset (HR-TO). PEAP was significantly higher in IDC patients compared to controls (48.7 +/- 32.6 vs. 9.8 +/- 5.4 %, P < 0.01), whereas MBP-TS was lower (0.97 +/- 0.60 vs. 2.07 +/- 1.04 mmHg BBI(-1) (BBI, beat-to-beat interval), P < 0.05), as was HR-TS (8.46 +/- 7.90 vs. 30.73 +/- 22.90 ms BBI(-1), P < 0.01). HR-TO was significantly higher in IDC patients (-0.56 +/- 2.19 vs. -5.52 +/- 4.13 %, P < 0.01). In addition, the regulation patterns of BP and HR following a single PVC differed significantly between IDC patients and controls. Specifically, we observed pronounced PEAPs in IDC patients. The baroreflex response initiated by the low pressure amplitude of the PVC was suppressed in IDC patients due to the augmented potentiation of the first postextrasystolic blood pressure. Furthermore, IDC patients displayed impressive postextrasystolic pulsus alternans phenomena, whereas healthy subjects exhibited a typical baroreflex pattern. The pulsus alternans phenomenon seems to be triggered by a PVC.