Virological Diagnosis of Central Nervous System Infections by Use of PCR Coupled with Mass Spectrometry Analysis of Cerebrospinal Fluid Samples

Viruses are the leading cause of central nervous system (CNS) infections, ahead of bacteria, parasites, and fungal agents. A rapid and comprehensive virologic diagnostic testing method is needed to improve the therapeutic management of hospitalized pediatric or adult patients. In this study, we assessed the clinical performance of PCR amplification coupled with electrospray ionization-time of flight mass spectrometry analysis (PCR-MS) for the diagnosis of viral CNS infections. Three hundred twenty-seven cerebrospinal fluid (CSF) samples prospectively tested by routine PCR assays between 2004 and 2012 in two university hospital centers (Toulouse and Reims, France) were retrospectively analyzed by PCR-MS analysis using primers targeted to adenovirus, human herpesviruses 1 to 8 (HHV-1 to -8), polyomaviruses BK and JC, parvovirus B19, and enteroviruses (EV). PCR-MS detected single or multiple virus infections in 190 (83%) of the 229 samples that tested positive by routine PCR analysis and in 10 (10.2%) of the 98 samples that tested negative. The PCR-MS results correlated well with herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), and EV detection by routine PCR assays (kappa values [95% confidence intervals], 0.80 [0.69 to 0.92], 0.85 [0.71 to 0.98], and 0.84 [0.78 to 0.90], respectively), whereas a weak correlation was observed with Epstein-Barr virus (EBV) (0.34 [0.10 to 0.58]). Twenty-six coinfections and 16 instances of uncommon neurotropic viruses (HHV-7 [n = 13], parvovirus B19 [n = 2], and adenovirus [n = 1]) were identified by the PCR-MS analysis, whereas only 4 coinfections had been prospectively evidenced using routine PCR assays (P < 0.01). In conclusion, our results demonstrated that PCR-MS analysis is a valuable tool to identify common neurotropic viruses in CSF (with, however, limitations that were identified regarding EBV and EV detection) and may be of major interest in better understanding the clinical impact of multiple or neglected viral neurological infections.     

Detection,Identification, and Distribution of Fungi in Bronchoalveolar Lavage Specimens by Use of Multilocus PCR Coupled with Electrospray Ionization/Mass Spectrometry

As pulmonary fungal infections continue to increase due to an increasing number of immunocompromised patients, rapid detection and accurate identification of these fungal pathogens are critical. A broad fungal assay was developed by incorporating broad-range multilocus PCR amplification and electrospray ionization/mass spectrometry (PCR/ESI-MS) to detect and identify fungal organisms directly from clinical specimens. The aims of this study were to evaluate the performance of PCR/ESI-MS for detection, identification, and determination of the distribution of fungal organisms in bronchoalveolar lavage (BAL) fluid specimens. The BAL fluid specimens submitted for fungal culture at Vanderbilt University Medical Center between May 2005 and October 2011 were included. Cultures and identification were done using standard procedures. In addition, DNA was extracted from BAL fluid specimens, and fungal DNA amplification/identification were performed by PCR/ESI-MS. The results were compared with those of the standard cultures. A total of 691 nonduplicated BAL fluid specimens with sufficient leftover volume for molecular testing were evaluated using PCR/ESI-MS. Among them, 134 specimens (19.4%) were positive for fungi by both culture and PCR/ESI-MS testing. Of the dual-positive specimens, 125 (93.3%) were positive for Candida and Aspergillus species, with concordances between culture and PCR/ESI-MS results being 84 (67.2%) at the species level and 109 (87.2%) at the genus level. In addition, 243 (35.2%) and 30 (4.3%) specimens were positive only by PCR/ESI-MS or by culture, respectively (odds ratio [OR] = 11.95, 95% confidence interval [CI] = 7.90 to 18.17, P = 0.0000). Codetection of fungal organisms was noted in 23 (3.3%) specimens by PCR/ESI-MS, which was significantly higher than the 4 (0.6%) in which they were noted by culture (OR = 5.91, 95% CI = 1.93 to 20.27, P = 0.0002). Among 53 specimens in which cultures failed because of bacterial overgrowth, at least one fungus was identified in 26 specimens (47.3%) by PCR/ESI-MS. PCR/ESI-MS provides an advanced tool for rapid and sensitive detection, identification, and determination of the distribution of fungal organisms directly from BAL fluid specimens. Moreover, it detected fungal organisms in specimens in which cultures failed because of bacterial overgrowth. The clinical relevance of the significantly higher detection rate of fungal organisms by PCR/ESI-MS merits further investigation.     

Rapid Identification of Bacteria and Candida Pathogens in Peritoneal Dialysis Effluent from Patients with Peritoneal Dialysis-Related Peritonitis by Use of Multilocus PCR Coupled with Electrospray Ionization Mass Spectrometry

PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was compared with culture for pathogen detection in peritoneal dialysis (PD)-related peritonitis. Of 21 samples of PD effluent, PCR/ESI-MS identified microorganisms in 18 (86%) samples, including Mycobacterium tuberculosis in 1 culture-negative sample. Of 15 double-positive samples, PCR/ESI-MS and culture reached levels of agreement of 100% (15/15) and 87.5% (7/8) at the genus and species levels, respectively. PCR/ESI-MS can be used for rapid pathogen detection in PD-related peritonitis.     

Determinants of Right Ventricular Muscle Mass in Idiopathic Dilated Cardiomyopathy: Impact of Left Ventricular Muscle Mass and Pulmonary Hypertension

Introduction: Although chronic pulmonary hypertension and right ventricular (RV) function carry important functional and prognostic implications in idiopathic dilated cardiomyopathy (IDC), little information on RV muscle mass (RVMM) and its determinants has been published.Methods: Our study comprised thirty-five consecutive patients with IDC, left ventricular (LV) ejection fraction <40% and NYHA class ≥2. Hemodynamic data and parameters on LV and RV geometry were derived from right heart catheterisation and cardiac magnetic resonance imaging.Results: RVMM was normalized to body size using a common linear, body surface area based approach (RVMMI) and by an allometric index (RVMM-AI) incorporating adjustment for age, height and weight. Stepwise multiple regression analysis revealed that pulmonary artery pressure and left ventricular muscle mass were independent predictors of RVMM-AI. The interventricular mass ratio of RV and LV mass (IVRM) was closely related to RVMM (r = 0.79, p < 0.001) and total muscle mass (r = 0.39, p < 0.02). However, there was no significant relationship between LVMM and IVMR (r = 0.17, p = 0.32).Conclusion: Our data suggest that an increase in RV mass in IDC may be explained by two mechanisms: First, as a consequence of the myopathic process itself resulting in a balanced hypertrophy of both ventricles. Second, due to the chamber specific burden of pulmonary artery pressure rise, resulting in unbalanced RV hypertrophy.     

PCR-Electrospray Ionization Mass Spectrometry for Direct Detection of Pathogens and Antimicrobial Resistance from Heart Valves in Patients with Infective Endocarditis

Microbiological diagnosis is pivotal to the appropriate management and treatment of infective endocarditis. We evaluated PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) for bacterial and candidal detection using 83 formalin-fixed paraffin-embedded heart valves from subjects with endocarditis who had positive valve and/or blood cultures, 63 of whom had positive valvular Gram stains. PCR/ESI-MS yielded 55% positivity with concordant microbiology at the genus/species or organism group level (e.g., viridans group streptococci), 11% positivity with discordant microbiology, and 34% with no detection. PCR/ESI-MS detected all antimicrobial resistance encoded by mecA or vanA/B and identified a case of Tropheryma whipplei endocarditis not previously recognized.     

Detection and Direct Typing of Herpes Simplex Virus in Perianal Ulcers of Patients with AIDS by PCR

The presence of herpes simplex virus type 1 (HSV-1) and HSV-2 in perianal ulcerations of 41 AIDS patients was assessed by virus culture and a type-specific PCR-based assay. HSV was isolated from the lesion site in 24 of 41 (58.5%) patients, and HSV DNA was detected by PCR in all 24 (100%) of these specimens. Additionally, PCR was used to detect HSV DNA in 12 of 17 (70.5%) HSV culture-negative samples. Thus, HSV genomic sequences could be demonstrated in 36 of 41 (87.8%) perianal ulcers in this series. Full agreement in HSV typing by either immunodot assay or PCR was seen in 24 samples that were positive by both virus culture and PCR. HSV-2 was demonstrated in 35 of 36 (97.2%) HSV-positive samples.     

扩张型心肌病患者Th17/Treg的变化及意义

目的:探讨Th17/Treg的变化及其与扩张型心肌病(DCM)发生发展的关系。方法:收集急性病毒性心肌炎(AVMC)患者及DCM患者和健康体检者的外周血,ELISA法检测血清中IL-17及TGF-β1浓度;经PE-CD4、FITC-CD25及FITC-IL-17单抗染色后,双色流式细胞术检测Th17细胞及Treg细胞的比例。结果:AVMC组Th17和IL-17高于DCM组和对照组;DCM组Treg和TGF-β1低于AVMC组与对照组;AVMC组与DCM组Th17/Treg无显著差异,但均高于对照组。结论:Thl7/Treg的升高打破了免疫平衡,促进了AVMC的发病及其向DCM的发展。     

电感耦合等离子体质谱法检测尿液中镍、铬含量

本文就使用电感耦合等离子体质谱仪同时测定尿液中镍、铬元素含量的方法进行了研究。样品经简单稀释后直接进样,采用动态反应池技术消除离子干扰,并使用内标进行校正。尿液中镍、铬元素的检出限分别为0.06μg/L和0.001μg/L,检测相对标准偏差在1.4%~4.7%间,加标回收率为99.1%~102.4%,同时质控样品值也落入可信参考范围。本方法样品处理简单,测量快速、灵敏、准确,值得临床推广应用。     

Detection and Identification of Yeasts from Formalin-Fixed,Paraffin-Embedded Tissue by Use of PCR-Electrospray Ionization Mass Spectrometry

Diagnosis of yeast infection is typically accomplished by fungal smear and culture, histopathologic examination, and/or serologic studies. Newer assays based on mass spectrometry may be useful for yeast identification when histologic examination is inconclusive, fungal cultures are not ordered, or cultures fail to yield a causative agent. The purpose of this study was to evaluate the ability of the PLEX-ID broad fungal assay to accurately detect and identify yeasts in formalin-fixed paraffin-embedded (FFPE) tissues. Tissue samples from 78 FFPE specimens with both histopathology and corresponding culture results for a variety of yeasts were tested using the PLEX-ID broad fungal assay. A 40-μm FFPE tissue section from each specimen was digested with proteinase K followed by nucleic acid extraction and PCR using broad-range fungal primers. Yeast DNA in amplified products was identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA gene sequencing. PLEX-ID analysis detected yeast DNA in 78.2% (61/78) of the cases, of which 91.8% (56/61) were concordant with culture results. Of the 5 discordant positive results, 4 PLEX-ID results were considered to result from environmental contaminants, while 1 clinically important discrepancy was observed (Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing of the discordant sample was unsuccessful. The majority of histopathology results (89.7% [70/78]) correlated with culture results. The PLEX-ID broad fungal assay identifies fungi directly from FFPE tissues and can be a useful adjunct to traditional culture and histopathology tests.     

Detection of Leptospira DNA in Patients with Aseptic Meningitis by PCR

Samples of cerebrospinal fluid from 103 patients with aseptic meningitis were tested by PCR for detection of leptospires, and the results were compared with those of the microscopic agglutination test (MAT) and an enzyme-linked immunosorbent assay for detection of immunoglobulin M (ELISA-IgM). Of these samples, 39.80% were positive by PCR and 8.74 and 3.88% were positive by MAT and ELISA-IgM, respectively.     

Specific Detection of Fusarium Species in Blood and Tissues by a PCR Technique

Fusarium species are opportunistic nosocomial pathogens that often cause fatal invasive mycoses. We designed a primer pair that amplifies by PCR a fragment of a gene coding for the rRNA of Fusarium species. The DNAs of the main Fusarium species and Neocosmospora vasinfecta but not the DNAs from 11 medically important fungi were amplified by these primers. The lower limit of detection of the PCR system was 10 fg of Fusarium solani DNA by ethidium bromide staining. To test the ability of this PCR system to detect Fusarium DNA in tissues, we developed a mouse model of disseminated fusariosis. Using the PCR, we detected Fusarium DNA in mouse tissues and in spiked human blood. Furthermore, F. solani, Fusarium moniliforme, and Fusarium oxysporum were testing by random amplified polymorphic DNA (RAPD) analysis. The bands produced by RAPD analysis were purified, cloned, and sequenced. The information was used to design primer pairs that selectively amplified one or several Fusarium species. The method developed may be useful for the rapid detection and identification of Fusarium species both from culture and from clinical samples.     

Detection of Prosthetic Joint Infection by Use of PCR-Electrospray Ionization Mass Spectrometry Applied to Synovial Fluid

PCR coupled with electrospray ionization mass spectrometry applied to synovial fluid specimens had an 81% sensitivity and a 95% specificity for the diagnosis of prosthetic joint infection.     

HLA-DQA1, -DQB1 Polymorphism and Genetic Susceptibility to Idiopathic Dilated Cardiomyopathy in Hans of Northern China

Autoimmune mechanisms are likely to participate in the pathogenesis of at least a subgroup of idiopathic dilated cardiomyopathy (IDC), and components of the major histocompatibility complex (MHC) may serve as markers for the propensity to develop immune‐mediated myocardial damage. Human leukocyte antigen (HLA) class II genes, especially HLA‐DQ genes, which are highly polymorphic, play an important role in the activation of immune responses and thus control the predisposition to, or protection from, IDC. This study was conducted to investigate the association of HLA‐DQA1, ‐DQB1 allele polymorphisms with an autoantibody against the myocardial mitochondria ADP/ATP carrier, and to explore susceptibility to idiopathic dilated cardiomyopathy (IDC) among the Han ethnic group in northern China and the immunological mechanisms and hereditary susceptibility to IDC. Polymerase chain reaction sequence‐specific primer (PCR‐SSP) techniques were used to analyze polymorphisms of the second exon of HLA‐DQA1 and ‐DQB1 alleles among 68 unrelated IDC patients, 4 probands of IDC pedigrees, and 100 healthy controls, all of Han nationality and having lived in northern China for a long time. Following echocardiography examination the IDC subjects were stratified according to ejection fraction (EF) values. Those with EF values higher than 50% were placed in subgroup 1, subgroup 2 included the patients with an EF value of 15–35%, and subgroup 3 consisted of those whose EF values were less than 15%. An autoantibody against the myocardial mitochondria ADP/ATP carrier was examined using immunoblot analysis. The frequencies of HLA‐DQA1*0501 and HLA‐DQB1*0303 were 0.3889 and 0.1806 in the IDC group, significantly higher than those of the healthy controls (0.0900 and 0.0364 respectively, both P < 0.05). The OR was 5.20 (95% CI: 3.60–8.50) and 4.85 (95% CI: 2.56–9.39) respectively. Further analysis of the three subgroups showed a higher frequency of HLA‐DQA1*0501 among patients whose EF was less than 15% than those whose EF values were ≥15%. Conversely, the frequencies of HLA‐DQA1*0201 and ‐DQB1*0502, *0504 were significantly lower in the IDC group (0.0139, 0.0139 and 0.0417 respectively) than in the control group (0.2000, 0.0727 and 0.1091 respectively) (P < 0.05). The frequency of the HLA‐DQA1*0501 allele was significantly higher in IDC patients whose autoantibody is positive in contrast with those whose autoantibody is negative (18.57% vs. 5.86%, P < 0.05); the relative risk (RR) was 4.32. The other frequencies of HLA‐DQA1 and ‐DQB1 alleles showed no significant difference in the antibody positive and negative groups of IDC patients. The alleles of HLA‐DQA1*0501 and HLA‐DQB1*0303 were closely associated with poor EF values in the IDC group, and may be involved in susceptibility to the disease. The DQA1*0201 and DQB1*0502, *0504 alleles may confer protection to IDC among individuals of northern Chinese Han nationality. The SER⁵⁷ residue in the second exon of DQB1*0502 and *0504 may confer resistance to IDC, and defects or substitution of this amino acid residue at position 57 of the DQβ chain may be associated with IDC susceptibility. HLA‐DQ allele polymorphisms may serve as genetic markers for IDC and be involved in the regulation of the immune specific response to auto or exterior anti‐myocardium antibodies.     

Non-invasive Model-Based Assessment of Passive Left-Ventricular Myocardial Stiffness in Healthy Subjects and in Patients with Non-ischemic Dilated Cardiomyopathy

Patient-specific modelling has emerged as a tool for studying heart function, demonstrating the potential to provide non-invasive estimates of tissue passive stiffness. However, reliable use of model-derived stiffness requires sufficient model accuracy and unique estimation of model parameters. In this paper we present personalised models of cardiac mechanics, focusing on improving model accuracy, while ensuring unique parametrisation. The influence of principal model uncertainties on accuracy and parameter identifiability was systematically assessed in a group of patients with dilated cardiomyopathy (\(n=3\)) and healthy volunteers (\(n=5\)). For all cases, we examined three circumferentially symmetric fibre distributions and two epicardial boundary conditions. Our results demonstrated the ability of data-derived boundary conditions to improve model accuracy and highlighted the influence of the assumed fibre distribution on both model fidelity and stiffness estimates. The model personalisation pipeline—based strictly on non-invasive data—produced unique parameter estimates and satisfactory model errors for all cases, supporting the selected model assumptions. The thorough analysis performed enabled the comparison of passive parameters between volunteers and dilated cardiomyopathy patients, illustrating elevated stiffness in diseased hearts.     

Rapid and Direct Detection of Herpes Simplex Virus in Cerebrospinal Fluid by Use of a Commercial Real-Time PCR Assay

Central nervous system infection due to herpes simplex virus (HSV) is a medical emergency and requires rapid diagnosis and initiation of therapy. In this study, we compared a routine real-time PCR assay for HSV types 1 (HSV-1) and 2 (HSV-2) to a recently FDA-approved direct PCR assay (Simplexa HSV-1/2 Direct; Focus Diagnostics, Cypress, CA) using cerebrospinal fluid samples (n = 100). The Simplexa HSV-1/2 assays demonstrated a combined sensitivity and specificity of 96.2% (50/52) and 97.9% (47/48), respectively. In addition, the Simplexa assay does not require nucleic acid extraction, and the results are available in 60 min.     

Rapid Identification of Biothreat and Other Clinically Relevant Bacterial Species by Use of Universal PCR Coupled with High-Resolution Melting Analysis

A rapid assay for eubacterial species identification is described using high-resolution melt analysis to characterize PCR products. Unique melt profiles generated from multiple hypervariable regions of the 16S rRNA gene for 100 clinically relevant bacterial pathogens, including category A and B biothreat agents and their surrogates, allowed highly specific species identification.Rapid and accurate diagnostic tools are critical for infectious disease surveillance and early diagnosis of disease (8, 12). A simple platform which could deliver broad-based screening and specific pathogen identification would be invaluable for the timely recognition of emerging and biothreat (BT) outbreaks, as well as commonly encountered clinical infections (2, 7, 9, 11, 12).We previously reported a probe-based PCR assay, which utilizes conserved and variable 16S rRNA gene sequences for initial broad-based eubacterial detection and subsequent identification of specific bacterial agents (11). The assay demonstrated high analytical sensitivity but was limited by an inability to differentiate closely related pathogens due to decreased specificity of the TaqMan probe chemistry and high sequence homology within selected hypervariable regions of the 16S rRNA gene. Probe-based amplicon characterization accordingly limits testing to a finite number of anticipated pathogens. Alternative strategies for amplicon analysis, such as sequencing and mass spectrometry, allow broader-scale product characterization but are costly, time-consuming, and lacking in throughput (1, 6). High-resolution melt analysis (HRMA) offers a simple, low-cost, closed-tube approach to amplicon analysis with the capacity for single-nucleotide discrimination and easy integration with PCR analysis (10). We report a unique strategy for the rapid, highly specific identification of BT- related and non-BT-related bacterial pathogens which couples eubacterial PCR with HRMA.Three hypervariable regions (V1, V3, and V6), each flanked by highly conserved sequences within the 16S rRNA gene, were selected for primer design (3). Sequence data for clinically or BT-relevant bacteria were obtained from GenBank and aligned using ClustalW (www.ebi.ac.uk/clustalw/) to determine sequence variability. Primer pairs used to target hypervariable regions were as follows: V1-F (5′-GYGGCGNACGGGTGAGTAA-3′) and V1-R (5′-TTACCCCACCAACTAGC-3′), V3-F (5′-CCAGACTCCTACGGGAGGCAG-3′) and V3-R (5′-CGTATTACCGCGGCTGCTG-3′), and V6-F (5′-TGGAGCATGTGGTTTAATTCGA-3′) and V6-R (5′-AGCTGACGACANCCATGCA-3′).One hundred common, BT-related, and BT-surrogate organisms composed of 58 different bacterial species of American Type Culture Collection (ATCC) strains, clinical isolates, or inactivated or nonpathogenic strains were used for analysis (Table ​(Table1).1). Ten to 15 colonies of each bacterial organism were inoculated in to 200 μl of molecular-grade water (Roche Molecular Diagnostics, Indianapolis, IN), and DNA was extracted using a Roche MAGNA Pure instrument (Roche Molecular Corporation, Indianapolis, IN). Archived DNA extracted as previously described from 40 archived clinical synovial fluid (14) and cerebral spinal fluid samples collected from patients suspected of having septic arthritis or bacterial meningitis, respectively, were also used for blinded analyses.

TABLE 1.

Melting analysis of non-BT-related and BT-related organisms

The Number of Regulatory T Cells Correlates with Hemodynamic Improvement in Patients with Inflammatory Dilated Cardiomyopathy After Immunoadsorption Therapy

Inflammatory DCM (iDCM) may be related to autoimmune processes. An immunoadsorption (IA) has been reported to improve cardiac hemodynamics. The benefit of IA is probably related to the removal of autoantibodies. A recent study suggests additional effects of IA on the T cell–mediated immune reactions, especially on regulatory T cells (Tregs). In this prospective study, the correlation between the level of Tregs and improvement of myocardial contractility in response to IA in patients with iDCM was investigated. Patients (n = 18) with iDCM, reduced left ventricular (LV) ejection fraction (<35%), were enrolled for IA. Before and 6 months after IA, LV systolic function was assessed by echocardiography, and blood levels of Tregs were quantified by FACS analysis. Patients (n = 12) with chronic ischaemic heart failure and comparable reduced LV‐EF served as controls. IA improved LV‐EF in 12 of 18 patients at 6‐month follow‐up. These patients were classified as ‘IA responder’. In 6 patients, LV‐EF remained unchanged. At baseline, IA responder and non‐responder subgroups showed similar values for C‐reactive protein, white blood cells, lymphocytes and T helper cells, but they differ for the number of circulating Tregs (responder: 2.32 ± 1.38% versus non‐responder: 4.86 ± 0.28%; P < 0.01). Tregs increased significantly in the IA responders, but remained unchanged in the IA non‐responders. In patients with ischaemic cardiomyopathy, none of these values changed over time. A low level of Tregs in patients with chronic iDCM may characterize a subset of patients who do best respond to IA therapy.